Preparing figures for publication --PPI vs DPI

[Page Baluch]

They become the middle man/insurance agent by negotiating a service agreement directly with the vendor company and the services provided are based on what is included in your current service contract. So a yearly maintenance visit is still included as well as the costs of repairs (including travel fees). Because of their volume they offer a 25% discount on the service contract cost. It appears to provide the same service you would have received originally but you may loose some perks because you may not have that direct relationship with the vendor (i.e. many of our techs check other stuff while they are there that may not be under warranty). They have listed some universities (for ex: Univ Alabama, Birmingham) that are switching most of their extended service plans to this system. These groups are having equipment from various departments join in as well as office equipment (i.e. printers) which in the end saves them lots of money. A few advantages I see is that it will offer some relief on over priced service contracts that are provided by small companies (we just acquired a multiphoton system that will cost us $43,000/yr in service contract fees!!) and through this system they are developing a bank of information regarding the quality of the instruments that are available on the market that their customers can access.

Page

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carl Boswell
Sent: Friday, July 09, 2010 9:50 AM
To: [hidden email]
Subject: Re: Service Contract underwriters

Hi Page,

Their website says: "THE REMI GROUP does not repair or maintain equipment...".  What does the service provide?  How is cost reduced?

 

Thanks,

c

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Friday, July 09, 2010 9:08 AM

Subject: Service Contract underwriters

At ASU we are looking into using underwriters, such as REMI, to cover our large equipment service contracts in order to cut down on costs. Does anyone have experience with using this type of service? Do you have any cautions or recommendations?

Thanks!

Page 

 

Page Baluch
W.M. Keck Lab
Arizona State University/SoLS

 

[Williams, Geoffrey]

From what I understand they don't negotiate a service agreement.

What they do is based on repair costs of the microscope they take a
gamble at how much it will cost to service the system in a year.

It works like insurance.  The company knows it isn't going to be
spending the full amount from all of their policies, if it did, they
wouldn't be in business.  So they spread the risk out.  It is to their
advantage to get as many microscopes covered as possible.

How service works (and this is the part I have issues with): you call
an 800 number give them the instrument number and tell them briefly
what the problem is.  They contact the service provider (for example
if you have a Leica SP2, then they call Leica) get a quote for the
problem and then issue a P.O.#.

Then once Leica has the PO # they can contact you to schedule the repair.

In general this can add a day or two and as much as a week between the
call for service and actually having someone on site fixing the
instrument.  For a low use system, like an older confocal that gets
used occasionally but has an outrageously expensive service contract,
the system works pretty well.   If you have a high use system and a
strong working relationship with your current service contract
people/engineers you may lose a significant amount of up time if you
have chronic problems.

And say with a PM, if it takes more than a day, they have to request
an extension to the PO.  This can happen pretty quickly, but still
means rather than starting and finishing the job with first priority,
the service engineer works until the initial PO is maxed out then
heads home and comes back a day or two or a week later to resume work.

Disclaimer:  We are doing a "trial" period with our least used
confocal microscope (SP2 with AOBS, 405, 4 PMT + 1 TPMT), and my
comments above are based on my limited personal experience with the
Underwriters.

It would never have worked to keep out Zeiss systems running.  Yes the
Zeiss require a bit more work but also a fair bit of direct phone
support.  It also gets used significantly more, if we had to wait for
a week before someone could come in and fix the system it would be
disastrous.

It makes sense for older scopes with very expensive factory contracts
that don't get enough use to generate enough recharge to cover the
costs and where you can afford to have it down for some periods of
time.

YMMV

Geoff Williams
Leduc Bioimaging Facility Manager
Brown University

http://www.brown.edu/Facilities/Leduc_Bioimaging_Facility/



On Fri, Jul 9, 2010 at 1:36 PM, Debra Baluch <[hidden email]> wrote:

> They become the middle man/insurance agent by negotiating a service
> agreement directly with the vendor company and the services provided are
> based on what is included in your current service contract. So a yearly
> maintenance visit is still included as well as the costs of repairs
> (including travel fees). Because of their volume they offer a 25% discount
> on the service contract cost. It appears to provide the same service you
> would have received originally but you may loose some perks because you may
> not have that direct relationship with the vendor (i.e. many of our
> techs check other stuff while they are there that may not be under
> warranty). They have listed some universities (for ex: Univ Alabama,
> Birmingham) that are switching most of their extended service plans to this
> system. These groups are having equipment from various departments join in
> as well as office equipment (i.e. printers) which in the end saves them lots
> of money. A few advantages I see is that it will offer some relief on over
> priced service contracts that are provided by small companies (we just
> acquired a multiphoton system that will cost us $43,000/yr in service
> contract fees!!) and through this system they are developing a bank of
> information regarding the quality of the instruments that are available on
> the market that their customers can access.
>
> Page
>
>
> ________________________________
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Carl Boswell
> Sent: Friday, July 09, 2010 9:50 AM
> To: [hidden email]
> Subject: Re: Service Contract underwriters
>
> Hi Page,
> Their website says: "THE REMI GROUP does not repair or maintain
> equipment...".  What does the service provide?  How is cost reduced?
>
> Thanks,
> c
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-954-7053
> FAX [phone]
>
> ----- Original Message -----
> From: Debra Baluch
> To: [hidden email]
> Sent: Friday, July 09, 2010 9:08 AM
> Subject: Service Contract underwriters
> At ASU we are looking into using underwriters, such as REMI, to cover our
> large equipment service contracts in order to cut down on costs. Does anyone
> have experience with using this type of service? Do you have any cautions or
> recommendations?
> Thanks!
> Page
>
>
> Page Baluch
> W.M. Keck Lab
> Arizona State University/SoLS
>
>
>

[George McNamara]

Hi Jason,

Please include spectra. PubSpectra is a single Excel 2007 file, XML, standardized format. The challenge for me is that spectra are published as graphs, not data. I occasionally look at chemical journals that have mass spec graphs - would be impossible to un-digitize. I doubt you'll get any love from the America Chemical Society on including all data as data, but the Royal Society of Chemistry is worth (your) reaching out to. Also to Peter Murray-Rust, e.g.

1. [PDF]

2. Open Data in Science

   
   
   File Format: PDF/Adobe Acrobat - Quick View by P Murray-Rust - 2008 - Cited by 6 - Related articles Jan 18, 2008 ... expose their SI as Open Data (the Royal Society of Chemistry is an .... has also developed a Protocol for Implementing Open Access Data. ... precedin
   
   
   

Sincerely,

George

At 03:41 PM 7/7/2010, you wrote:

Dear All-

...

That is certainly one avenue.  We are trying many ways-- we want to extend the DataViewer as soon as we can:  more types of data, more journals, and more functionality.
 

Great work JCB and Jason et al!

Thanks for your support.

Cheers,

Jason
 
--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee  DD1 5EH
United Kingdom

phone [phone]
Intl phone:  [phone]
FAX   [phone]
email: [hidden email]

Lab Page: http://gre.lifesci.dundee.ac.uk/staff/jason_swedlow.html
Open Microscopy Environment: http://openmicroscopy.org
**************************

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[George McNamara]

Hi Carl,

This is a big risk because the vendor, i.e. Zeiss, Leica, et al, may put you at the end of the line for service calls. The flow cytometry core here had some BD flow cytometers under a "Fisher contract" (I am not sure whether this is the Fisher in Thermo Fisher), and BD policy was that if the underwritten instrument was in need of a service call, if the BD service tech had not reached the door of the underwritten instrument lab, they would be diverted to a full paying customer if need arose. Apparently there were enough BD flow cytometers/sorters in the south Florida service territory that the underwritten instrument was often down and not serviced.

The purchasing dept people push these contracts because it makes them look good (I won't speculate here about other possible financial renumeration to them), and they are not the one's affected by instrument down time.

George


At 12:49 PM 7/9/2010, you wrote:

Hi Page,
Their website says: "THE REMI GROUP does not repair or maintain equipment...".  What does the service provide?  How is cost reduced?
 
Thanks,
c
 
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Friday, July 09, 2010 9:08 AM

Subject: Service Contract underwriters

At ASU we are looking into using underwriters, such as REMI, to cover our large equipment service contracts in order to cut down on costs. Does anyone have experience with using this type of service? Do you have any cautions or recommendations?

Thanks!

Page

 

Page Baluch

W.M. Keck Lab

Arizona State University/SoLS

 

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

[Jason Swedlow]

Hi George-

Interesting, this was exactly what we wanted to do originally with OME-XML-- include real data that define the experiment.  After some consultation, we elected to remove the filter spectra as, at that point, they didn't seem that useful-- you couldn't get spectra in OD's, and there was not a standard for the data (as you now refer to), and the spectra were becoming easily available on the web.  We did add and keep the objective OTF, since we felt that this should be stored with the image (we know of no examples of this being used unfortunately).  

We'll discuss it.  I suspect our strategy will be to drive adoption at the level we have, and then move stepwise towards the completeness you suggest.  But thanks for pointing this out-- very important.

Cheers,

Jason

On Sat, Jul 10, 2010 at 4:52 PM, George McNamara <[hidden email]> wrote:

Hi Jason,

Please include spectra. PubSpectra is a single Excel 2007 file, XML, standardized format. The challenge for me is that spectra are published as graphs, not data. I occasionally look at chemical journals that have mass spec graphs - would be impossible to un-digitize. I doubt you'll get any love from the America Chemical Society on including all data as data, but the Royal Society of Chemistry is worth (your) reaching out to. Also to Peter Murray-Rust, e.g.

1. [PDF]

2. Open Data in Science

   
   
   File Format: PDF/Adobe Acrobat - Quick View by P Murray-Rust - 2008 - Cited by 6 - Related articles Jan 18, 2008 ... expose their SI as Open Data (the Royal Society of Chemistry is an .... has also developed a Protocol for Implementing Open Access Data. ... precedin
   
   
   

Sincerely,

George

At 03:41 PM 7/7/2010, you wrote:

Dear All-

...

That is certainly one avenue.  We are trying many ways-- we want to extend the DataViewer as soon as we can:  more types of data, more journals, and more functionality.
 

Great work JCB and Jason et al!

Thanks for your support.

Cheers,

Jason
 
--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee  DD1 5EH
United Kingdom

phone [phone]
Intl phone:  [phone]
FAX   [phone]
email: [hidden email]

Lab Page: http://gre.lifesci.dundee.ac.uk/staff/jason_swedlow.html
Open Microscopy Environment: http://openmicroscopy.org
**************************

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara

--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee  DD1 5EH
United Kingdom

phone [phone]
Intl phone:  [phone]
FAX   [phone]
email: [hidden email]

Lab Page: http://gre.lifesci.dundee.ac.uk/staff/jason_swedlow.html
Open Microscopy Environment: http://openmicroscopy.org
**************************

[Jerry (Gerald) Sedgewick]

Hi All,

I'm a little late in addressing this issue, but the PPI/DPI part of this conversation is misleading.  What I mean to say is that the real issue may not be resolution, but reproduction.  Images that are sent to publishers with the full dynamic range of 0 - 255 pixel values may likely be reproduced with no details at the bright end (240 - 255), and the dark end (0 - 20).  Printing presses cannot reproduce detail within these tonal ranges because of limitations with dropping ink on paper without A) having the drop not stick when the tonal values are bright and B) having the inks blend into each other through capillary action at the darkest values.  This phenomenon gets worse when the paper is lower quality (e.g., "Science").

Color reproduction is generally worse because primary and secondary colors are used to show experimental evidence.  These colors are not always within the range of printing presses, and so these tend to print blobs sans detail when color choices are not appropriate for publishing onto paper.

More often than not, the issue of whether or not details can be resolved by eye on a printed page is not that of how many pixels exist in the submitted image, but in how effectively tones and colors were fitted to the printing press.  This is especially true if the pdf image at non-zoomed, computer screen resolution reveals desired details, but the printed page does not:  it is more likely that a 133 line per inch screened image appearing in publication has more resolution than the computer screen (often figured at an average of 90 pixels per inch, with 72 pixels being the "old" standard).  The color and tonal range of reproduction of a computer screen is greater than on a printing press.

Like others before this email, I believe that it is best that scientists take the task of reproduction as much in their own hands as possible so that the outcome can be controlled.  The image is a reproduction of what was once under a microscope, and it behooves the researcher to make that appear as close to the original representation as possible. 

As far as images being data points, this also is true, and these are to remain unaltered (unless flatfield correcting, background subtracting, etc) for measuring.  A faithful representation of that image when reproduced is another matter altogether.

Cheers,

Jerry Sedgewick

[Daniel James White]

Dear Jerry,

cheers for adding your valuable input to this discussion.
Its been a long and interesting one so far.

On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Mon, 12 Jul 2010 12:47:51 -0500
> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
> <html>
> <head>
>  <meta content="text/html; charset=windows-1252"
> http-equiv="Content-Type">
> </head>
> <body bgcolor="#ffffff" text="#000000">
> Hi All,<br>
> <br>

maybe you might like to turn off HTML text formatting in your email client when you post to the lists
in this case its not so distracting but sometimes emails to this an other list as unreadable in digest mode
due to the thousands of html tags.


> I'm a little late in addressing this issue, but the PPI/DPI part of
> this conversation is misleading.  What I mean to say is that the real
> issue may not be resolution, but reproduction.  

A very good point.

> Images that are sent to
> publishers with the full dynamic range of 0 - 255 pixel values may
> likely be reproduced with no details at the bright end (240 - 255), and
> the dark end (0 - 20).  Printing presses cannot reproduce detail within
> these tonal ranges because of limitations with dropping ink on paper
> without A) having the drop not stick when the tonal values are bright
> and B) having the inks blend into each other through capillary action
> at the darkest values.  This phenomenon gets worse when the paper is
> lower quality (e.g., "Science").<br>

So what we are saying is maybe summarised as follows:

Small thumbnail images in print can only every be just that - pointers to go and look at the real digital image on some online source.
No one can hope to reach quantitative conclusions from a small print image.

We can probably consign print images on paper to the heap of old technologies,
which are no longer relevant to the work we do.

Images in PDF files "could" be useful, but not if they are lossy compressed and missing meta data, which they usually are.



> <br>
> Color reproduction is generally worse because primary and secondary
> colors are used to show experimental evidence.  These colors are not
> always within the range of printing presses, and so these tend to print
> blobs sans detail when color choices are not appropriate for publishing
> onto paper.<br>

To make matters worse....
fors most peoples eyes, blue is much fainter than red and green, with green often being precieved as brightest.
So forget even semi  quantitative comparisons of intensity there.

In any case we dont print in red green and blue, so these are silly choices for a print version of figures.
The print colour space would be better, Magenta, yellow and cyan.... but still for our quantitative purposed not good.

Screen (LCD/CRT) colours are more similar to out eyes trichromatic response.
That why they are red green and blue... but the blue looks darker then green problem still persists.

> <br>
> More often than not, the issue of whether or not details can be
> resolved by eye on a printed page is not that of how many pixels exist
> in the submitted image, but in how effectively tones and colors were
> fitted to the printing press.  This is especially true if the pdf image
> at non-zoomed, computer screen resolution reveals desired details, but
> the printed page does not:  it is more likely that a 133 line per inch
> screened image appearing in publication has more resolution than the
> computer screen (often figured at an average of 90 pixels per inch,
> with 72 pixels being the "old" standard).  The color and tonal range of
> reproduction of a computer screen is greater than on a printing press.<br>
> <br>

Indeed it is, and so is a better visualisation tool...
but to get the most out of it, we also need to be smart.
We cant compare the brightness of blue and green due to our physiology,
even if the screen is properly calibrated (usually not the case).

So, we should not ry to do that on screen or in print.

Our eyes are much better at discriminating greyscale brightness scale,
but actually we are sill pretty bad at that.

see this example of where we are easily fooled when trying to compare grey scale brightness.
http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html

The situation with colouyr is more complicated, and also beset with pitfalle
due to the way our optical system works... it tries to find contrast in any scene,
and even generates contrast or colour differences that are not there (but gave us an evolutionary advantage in picking fruit)

See the spiral image at the bottom f this colocalisation tutorial,
which shows us that we should be careful when interpreting colour merge multi channel images.

http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis

you can draw the spiral pattern yourself in imageJ with the marco script there,
and prove to yourself that your eyes lie to you.

Greyscale images can be made interpretable in a semi quantitative manner by using for instance the fire colour look up table.
Each bright colour represents some intensity value, so its easier to compare intensities over and between images.
Physicists and chemists do this all the time, and think we are mad for showing DAPI staining  in black to Blue - because "thats what it looks like"
They are right , we are mad to do that.


> Like others before this email, I believe that it is best that
> scientists take the task of reproduction as much in their own hands as
> possible so that the outcome can be controlled.  The image is a
> reproduction of what was once under a microscope, and it behooves the
> researcher to make that appear as close to the original representation
> as possible.  <br>

I'm not sure that this is ever going to be a helpful way to approach the problem.
No matter how carefully you set out your images and send them to the publisher,
you have no control and what they do with them next. None.

The only way around this is to have the images in the paper act only as thumbnails
which point to the original image data file(s) on an online repository,
like the JCB image viewer or our wished for public biological image database...

An image is not the sample, it always contain much less info that the sample did.
The trick is to keep the useful info as it passes through the scope lenses,
the detector, and the computer you your brain/eyes.
I agree, an image is a representation of the info that made it through the microscope to you.
Trying to represent it "faithfully" and as  it "truly looks" are both missing the mark.
The image is usually degraded by the blur of the Point spread function / OTF  and by various sources of noise.
Thus, the image is an artifact in of itself. What we want to know about is the sample.
The images contains info from the sample in a degraded  and incomplete from.
We have to work around that, and not pretend that the image fully represents the sample.
It does not.

> <br>
> As far as images being data points, this also is true, and these are to
> remain unaltered (unless flatfield correcting, background subtracting,
> etc) for measuring.  A faithful representation of that image when
> reproduced is another matter altogether.<br>

Indeed it is, and i think there is even no such thing as a faithful representation of the Sample as an image,
so the faithful representation of the image is also then something to think about.

I think what matters is How you represent the image data....
in some cases an illustration like Hooke's drawing of cells might even be better than a digital representation,
if you want to get a certain message across to the reader. Original digital image data available too of course online.  

cheers

Dan


 
> <br>
> Cheers,<br>
> <br>
> Jerry Sedgewick<br>

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net  BioImageXD
http://pacific.mpi-cbg.de                Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Dresden Imaging Facility Network
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Andreas Bruckbauer]

Dear all,
so far this discussion mainly focusses on "the  image" but one image alone can not represent the data. Think about the data set  represented by a distribution  which might be gaussian, so you would  need  at  least the mean and  the  width of the  distribution. What i want to say is that you will need to do a quantification and then show  an image  representing  the mean and show a histogram which gives the reader a clue about how the data  is  distributed. There might be more than  one variable which is  important for the  analysis and of  course you should do an idependend repeat of the  experiment.
So what would you suggest, sending all the original image files to the  data bank?
I still think that rather than re-analysing other scientists data a repeat of the  experiment in another lab is more important, there are a lot of thinks apart  from data analysis and representation which can go wrong. However i see the point that when the paper is about image analysis to  provide the original files to let other groups  repreat the analysis. 

best wishes

Andreas

-----Original Message-----
From: Daniel James White <[hidden email]>
To: [hidden email]
Sent: Tue, 13 Jul 2010 9:01
Subject: Re: Preparing figures for publication --PPI vs DPI

Dear Jerry,





cheers for adding your valuable input to this discussion. 


Its been a long and interesting one so far. 





On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:





> Date:    Mon, 12 Jul 2010 12:47:51 -0500


> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>


> Subject: Re: Preparing figures for publication --PPI vs DPI


> 


> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">


> <html>


> <head>


>  <meta content="text/html; charset=windows-1252"


> http-equiv="Content-Type">


> </head>


> <body bgcolor="#ffffff" text="#000000">


> Hi All,<br>


> <br>





maybe you might like to turn off HTML text formatting in your email client when 


you post to the lists


in this case its not so distracting but sometimes emails to this an other list 


as unreadable in digest mode


due to the thousands of html tags. 








> I'm a little late in addressing this issue, but the PPI/DPI part of


> this conversation is misleading.  What I mean to say is that the real


> issue may not be resolution, but reproduction.  





A very good point. 





> Images that are sent to


> publishers with the full dynamic range of 0 - 255 pixel values may


> likely be reproduced with no details at the bright end (240 - 255), and


> the dark end (0 - 20).  Printing presses cannot reproduce detail within


> these tonal ranges because of limitations with dropping ink on paper


> without A) having the drop not stick when the tonal values are bright


> and B) having the inks blend into each other through capillary action


> at the darkest values.  This phenomenon gets worse when the paper is


> lower quality (e.g., "Science").<br>





So what we are saying is maybe summarised as follows:





Small thumbnail images in print can only every be just that - pointers to go and 


look at the real digital image on some online source. 


No one can hope to reach quantitative conclusions from a small print image. 





We can probably consign print images on paper to the heap of old technologies, 


which are no longer relevant to the work we do. 





Images in PDF files "could" be useful, but not if they are lossy compressed and 


missing meta data, which they usually are. 











> <br>


> Color reproduction is generally worse because primary and secondary


> colors are used to show experimental evidence.  These colors are not


> always within the range of printing presses, and so these tend to print


> blobs sans detail when color choices are not appropriate for publishing


> onto paper.<br>





To make matters worse....


fors most peoples eyes, blue is much fainter than red and green, with green 


often being precieved as brightest. 


So forget even semi  quantitative comparisons of intensity there. 





In any case we dont print in red green and blue, so these are silly choices for 


a print version of figures. 


The print colour space would be better, Magenta, yellow and cyan.... but still 


for our quantitative purposed not good. 





Screen (LCD/CRT) colours are more similar to out eyes trichromatic response. 


That why they are red green and blue... but the blue looks darker then green 


problem still persists. 





> <br>


> More often than not, the issue of whether or not details can be


> resolved by eye on a printed page is not that of how many pixels exist


> in the submitted image, but in how effectively tones and colors were


> fitted to the printing press.  This is especially true if the pdf image


> at non-zoomed, computer screen resolution reveals desired details, but


> the printed page does not:  it is more likely that a 133 line per inch


> screened image appearing in publication has more resolution than the


> computer screen (often figured at an average of 90 pixels per inch,


> with 72 pixels being the "old" standard).  The color and tonal range of


> reproduction of a computer screen is greater than on a printing press.<br>


> <br>





Indeed it is, and so is a better visualisation tool...


but to get the most out of it, we also need to be smart. 


We cant compare the brightness of blue and green due to our physiology,


even if the screen is properly calibrated (usually not the case).





So, we should not ry to do that on screen or in print. 





Our eyes are much better at discriminating greyscale brightness scale, 


but actually we are sill pretty bad at that. 





see this example of where we are easily fooled when trying to compare grey scale 


brightness. 


http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html





The situation with colouyr is more complicated, and also beset with pitfalle


due to the way our optical system works... it tries to find contrast in any 


scene, 


and even generates contrast or colour differences that are not there (but gave 


us an evolutionary advantage in picking fruit)





See the spiral image at the bottom f this colocalisation tutorial,


which shows us that we should be careful when interpreting colour merge multi 


channel images. 





http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis





you can draw the spiral pattern yourself in imageJ with the marco script there, 


and prove to yourself that your eyes lie to you. 





Greyscale images can be made interpretable in a semi quantitative manner by 


using for instance the fire colour look up table.


Each bright colour represents some intensity value, so its easier to compare 


intensities over and between images. 


Physicists and chemists do this all the time, and think we are mad for showing 


DAPI staining  in black to Blue - because "thats what it looks like"


They are right , we are mad to do that. 








> Like others before this email, I believe that it is best that


> scientists take the task of reproduction as much in their own hands as


> possible so that the outcome can be controlled.  The image is a


> reproduction of what was once under a microscope, and it behooves the


> researcher to make that appear as close to the original representation


> as possible.  <br>





I'm not sure that this is ever going to be a helpful way to approach the 


problem. 


No matter how carefully you set out your images and send them to the publisher, 


you have no control and what they do with them next. None. 





The only way around this is to have the images in the paper act only as 


thumbnails


which point to the original image data file(s) on an online repository,


like the JCB image viewer or our wished for public biological image database...





An image is not the sample, it always contain much less info that the sample 


did. 


The trick is to keep the useful info as it passes through the scope lenses, 


the detector, and the computer you your brain/eyes. 


I agree, an image is a representation of the info that made it through the 


microscope to you. 


Trying to represent it "faithfully" and as  it "truly looks" are both missing 


the mark. 


The image is usually degraded by the blur of the Point spread function / OTF  


and by various sources of noise. 


Thus, the image is an artifact in of itself. What we want to know about is the 


sample. 


The images contains info from the sample in a degraded  and incomplete from. 


We have to work around that, and not pretend that the image fully represents the 


sample. 


It does not. 





> <br>


> As far as images being data points, this also is true, and these are to


> remain unaltered (unless flatfield correcting, background subtracting,


> etc) for measuring.  A faithful representation of that image when


> reproduced is another matter altogether.<br>





Indeed it is, and i think there is even no such thing as a faithful 


representation of the Sample as an image, 


so the faithful representation of the image is also then something to think 


about. 





I think what matters is How you represent the image data....


in some cases an illustration like Hooke's drawing of cells might even be better 


than a digital representation,


if you want to get a certain message across to the reader. Original digital 


image data available too of course online.  





cheers





Dan








 


> <br>


> Cheers,<br>


> <br>


> Jerry Sedgewick<br>





Dr. Daniel James White BSc. (Hons.) PhD


Senior Microscopist / Image Visualisation, Processing and Analysis


Light Microscopy and Image Processing Facilities 


Max Planck Institute of Molecular Cell Biology and Genetics


Pfotenhauerstrasse 108


01307 DRESDEN


Germany





[phone] (German Mobile)


[phone] (Work phone at MPI-CBG)


[phone] (Fax MPI-CBG LMF)





http://www.bioimagexd.net   BioImageXD


http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries Included)


http://www.chalkie.org.uk       Dan's Homepages


https://ifn.mpi-cbg.de          Dresden Imaging Facility Network


dan (at) chalkie.org.uk


( white (at) mpi-cbg.de )

[Armstrong, Brian]

Well, as George McNamara has suggested many times on the list the image you should send in the “mean image”, (an example of the images collected).

I would predict that this is rarely the case and that most images sent to journals are extreme examples of the best possible image collected and corrected.

However, sending the entire image set (in Gigabytes or even Terabytes) to the journal seems impractical. Perhaps instead one could acquire the entire data set by e-mailing the author.   

 

 

Brian D Armstrong PhD

Light Microscopy Core Manager

Beckman Research Institute

City of Hope

Dept of Neuroscience

1450 E Duarte Rd

Duarte, CA 91010

626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer
Sent: Tuesday, July 13, 2010 2:14 AM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

 

Dear all,
so far this discussion mainly focusses on "the  image" but one image alone can not represent the data. Think about the data set  represented by a distribution  which might be gaussian, so you would  need  at  least the mean and  the  width of the  distribution. What i want to say is that you will need to do a quantification and then show  an image  representing  the mean and show a histogram which gives the reader a clue about how the data  is  distributed. There might be more than  one variable which is  important for the  analysis and of  course you should do an idependend repeat of the  experiment.
So what would you suggest, sending all the original image files to the  data bank?
I still think that rather than re-analysing other scientists data a repeat of the  experiment in another lab is more important, there are a lot of thinks apart  from data analysis and representation which can go wrong. However i see the point that when the paper is about image analysis to  provide the original files to let other groups  repreat the analysis. 

best wishes

Andreas

 

 

 

-----Original Message-----
From: Daniel James White <[hidden email]>
To: [hidden email]
Sent: Tue, 13 Jul 2010 9:01
Subject: Re: Preparing figures for publication --PPI vs DPI

Dear Jerry,

cheers for adding your valuable input to this discussion. 

Its been a long and interesting one so far. 

On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:

> Date:    Mon, 12 Jul 2010 12:47:51 -0500

> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>

> Subject: Re: Preparing figures for publication --PPI vs DPI

> 

> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">

> <html>

> <head>

>  <meta content="text/html; charset=windows-1252"

> http-equiv="Content-Type">

> </head>

> <body bgcolor="#ffffff" text="#000000">

> Hi All,<br>

> <br>

maybe you might like to turn off HTML text formatting in your email client when 

you post to the lists

in this case its not so distracting but sometimes emails to this an other list 

as unreadable in digest mode

due to the thousands of html tags. 

> I'm a little late in addressing this issue, but the PPI/DPI part of

> this conversation is misleading.  What I mean to say is that the real

> issue may not be resolution, but reproduction.  

A very good point. 

> Images that are sent to

> publishers with the full dynamic range of 0 - 255 pixel values may

> likely be reproduced with no details at the bright end (240 - 255), and

> the dark end (0 - 20).  Printing presses cannot reproduce detail within

> these tonal ranges because of limitations with dropping ink on paper

> without A) having the drop not stick when the tonal values are bright

> and B) having the inks blend into each other through capillary action

> at the darkest values.  This phenomenon gets worse when the paper is

> lower quality (e.g., "Science").<br>

So what we are saying is maybe summarised as follows:

Small thumbnail images in print can only every be just that - pointers to go and 

look at the real digital image on some online source. 

No one can hope to reach quantitative conclusions from a small print image. 

We can probably consign print images on paper to the heap of old technologies, 

which are no longer relevant to the work we do. 

Images in PDF files "could" be useful, but not if they are lossy compressed and 

missing meta data, which they usually are. 

> <br>

> Color reproduction is generally worse because primary and secondary

> colors are used to show experimental evidence.  These colors are not

> always within the range of printing presses, and so these tend to print

> blobs sans detail when color choices are not appropriate for publishing

> onto paper.<br>

To make matters worse....

fors most peoples eyes, blue is much fainter than red and green, with green 

often being precieved as brightest. 

So forget even semi  quantitative comparisons of intensity there. 

In any case we dont print in red green and blue, so these are silly choices for 

a print version of figures. 

The print colour space would be better, Magenta, yellow and cyan.... but still 

for our quantitative purposed not good. 

Screen (LCD/CRT) colours are more similar to out eyes trichromatic response. 

That why they are red green and blue... but the blue looks darker then green 

problem still persists. 

> <br>

> More often than not, the issue of whether or not details can be

> resolved by eye on a printed page is not that of how many pixels exist

> in the submitted image, but in how effectively tones and colors were

> fitted to the printing press.  This is especially true if the pdf image

> at non-zoomed, computer screen resolution reveals desired details, but

> the printed page does not:  it is more likely that a 133 line per inch

> screened image appearing in publication has more resolution than the

> computer screen (often figured at an average of 90 pixels per inch,

> with 72 pixels being the "old" standard).  The color and tonal range of

> reproduction of a computer screen is greater than on a printing press.<br>

> <br>

Indeed it is, and so is a better visualisation tool...

but to get the most out of it, we also need to be smart. 

We cant compare the brightness of blue and green due to our physiology,

even if the screen is properly calibrated (usually not the case).

So, we should not ry to do that on screen or in print. 

Our eyes are much better at discriminating greyscale brightness scale, 

but actually we are sill pretty bad at that. 

see this example of where we are easily fooled when trying to compare grey scale 

brightness. 

http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html

The situation with colouyr is more complicated, and also beset with pitfalle

due to the way our optical system works... it tries to find contrast in any 

scene, 

and even generates contrast or colour differences that are not there (but gave 

us an evolutionary advantage in picking fruit)

See the spiral image at the bottom f this colocalisation tutorial,

which shows us that we should be careful when interpreting colour merge multi 

channel images. 

http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis

you can draw the spiral pattern yourself in imageJ with the marco script there, 

and prove to yourself that your eyes lie to you. 

Greyscale images can be made interpretable in a semi quantitative manner by 

using for instance the fire colour look up table.

Each bright colour represents some intensity value, so its easier to compare 

intensities over and between images. 

Physicists and chemists do this all the time, and think we are mad for showing 

DAPI staining  in black to Blue - because "thats what it looks like"

They are right , we are mad to do that. 

> Like others before this email, I believe that it is best that

> scientists take the task of reproduction as much in their own hands as

> possible so that the outcome can be controlled.  The image is a

> reproduction of what was once under a microscope, and it behooves the

> researcher to make that appear as close to the original representation

> as possible.  <br>

I'm not sure that this is ever going to be a helpful way to approach the 

problem. 

No matter how carefully you set out your images and send them to the publisher, 

you have no control and what they do with them next. None. 

The only way around this is to have the images in the paper act only as 

thumbnails

which point to the original image data file(s) on an online repository,

like the JCB image viewer or our wished for public biological image database...

An image is not the sample, it always contain much less info that the sample 

did. 

The trick is to keep the useful info as it passes through the scope lenses, 

the detector, and the computer you your brain/eyes. 

I agree, an image is a representation of the info that made it through the 

microscope to you. 

Trying to represent it "faithfully" and as  it "truly looks" are both missing 

the mark. 

The image is usually degraded by the blur of the Point spread function / OTF  

and by various sources of noise. 

Thus, the image is an artifact in of itself. What we want to know about is the 

sample. 

The images contains info from the sample in a degraded  and incomplete from. 

We have to work around that, and not pretend that the image fully represents the 

sample. 

It does not. 

> <br>

> As far as images being data points, this also is true, and these are to

> remain unaltered (unless flatfield correcting, background subtracting,

> etc) for measuring.  A faithful representation of that image when

> reproduced is another matter altogether.<br>

Indeed it is, and i think there is even no such thing as a faithful 

representation of the Sample as an image, 

so the faithful representation of the image is also then something to think 

about. 

I think what matters is How you represent the image data....

in some cases an illustration like Hooke's drawing of cells might even be better 

than a digital representation,

if you want to get a certain message across to the reader. Original digital 

image data available too of course online.  

cheers

Dan

> <br>

> Cheers,<br>

> <br>

> Jerry Sedgewick<br>

Dr. Daniel James White BSc. (Hons.) PhD

Senior Microscopist / Image Visualisation, Processing and Analysis

Light Microscopy and Image Processing Facilities 

Max Planck Institute of Molecular Cell Biology and Genetics

Pfotenhauerstrasse 108

01307 DRESDEN

Germany

[phone] (German Mobile)

[phone] (Work phone at MPI-CBG)

[phone] (Fax MPI-CBG LMF)

http://www.bioimagexd.net   BioImageXD

http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries Included)

http://www.chalkie.org.uk       Dan's Homepages

https://ifn.mpi-cbg.de          Dresden Imaging Facility Network

dan (at) chalkie.org.uk

( white (at) mpi-cbg.de )

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[Knecht, David]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

I have been forced back into this issue in helping someone get an image taken on our microscopes through a journal editor.  The images were fairly low resolution.  They were manipulated in Photoshop, imported into Canvas, text added and then output for the journal as a figure at 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when the images were indexed color, the up sampling was done by taking each pixel and subdividing it into smaller pixels.  Thus the pixelation of the original was maintained and the editor did not like that.  What they wanted done was to convert the images to RGB and then upsample.  In that case (or grayscale), Photoshop does an interpolation making a smoother looking zoomed in view, but that is a change in the data.  The index version is actually more accurate although less pleasing. I plan to argue that this is not necessary or desirable, but I did not know that Photoshop (but not ImageJ) makes this distinction and thought others might want to know it happens.  Does anyone know why index vs. RGB should matter to the up sampling algorithm.  THanks- Dave


On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:

> Well, as George McNamara has suggested many times on the list the image you should send in the “mean image”, (an example of the images collected).
> I would predict that this is rarely the case and that most images sent to journals are extreme examples of the best possible image collected and corrected.
> However, sending the entire image set (in Gigabytes or even Terabytes) to the journal seems impractical. Perhaps instead one could acquire the entire data set by e-mailing the author.  
>  
>  
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer
> Sent: Tuesday, July 13, 2010 2:14 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>  
> Dear all,
> so far this discussion mainly focusses on "the  image" but one image alone can not represent the data. Think about the data set  represented by a distribution  which might be gaussian, so you would  need  at  least the mean and  the  width of the  distribution. What i want to say is that you will need to do a quantification and then show  an image  representing  the mean and show a histogram which gives the reader a clue about how the data  is  distributed. There might be more than  one variable which is  important for the  analysis and of  course you should do an idependend repeat of the  experiment.
> So what would you suggest, sending all the original image files to the  data bank?
> I still think that rather than re-analysing other scientists data a repeat of the  experiment in another lab is more important, there are a lot of thinks apart  from data analysis and representation which can go wrong. However i see the point that when the paper is about image analysis to  provide the original files to let other groups  repreat the analysis.  
>
> best wishes
>
> Andreas
>
>  
>  
>  
> -----Original Message-----
> From: Daniel James White <[hidden email]>
> To: [hidden email]
> Sent: Tue, 13 Jul 2010 9:01
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear Jerry,
>
>
>
>  
>
>
>
>
>  
> cheers for adding your valuable input to this discussion.
>
>
>
>  
> Its been a long and interesting one so far.
>
>
>
>  
>
>
>
>
>  
> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest system wrote:
>
>
>
>  
>
>
>
>
>  
> > Date:    Mon, 12 Jul 2010 12:47:51 -0500
>
>
>
>  
> > From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>
>
>
>  
> > Subject: Re: Preparing figures for publication --PPI vs DPI
>
>
>
>  
> >
>
>
>
>  
> > <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>
>
>
>  
> > <html>
>
>
>
>  
> > <head>
>
>
>
>  
> >  <meta content="text/html; charset=windows-1252"
>
>
>
>  
> > http-equiv="Content-Type">
>
>
>
>  
> > </head>
>
>
>
>  
> > <body bgcolor="#ffffff" text="#000000">
>
>
>
>  
> > Hi All,<br>
>
>
>
>  
> > <br>
>
>
>
>  
>
>
>
>
>  
> maybe you might like to turn off HTML text formatting in your email client when
>
>
>
>  
> you post to the lists
>
>
>
>  
> in this case its not so distracting but sometimes emails to this an other list
>
>
>
>  
> as unreadable in digest mode
>
>
>
>  
> due to the thousands of html tags.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > I'm a little late in addressing this issue, but the PPI/DPI part of
>
>
>
>  
> > this conversation is misleading.  What I mean to say is that the real
>
>
>
>  
> > issue may not be resolution, but reproduction.  
>
>
>
>  
>
>
>
>
>  
> A very good point.
>
>
>
>  
>
>
>
>
>  
> > Images that are sent to
>
>
>
>  
> > publishers with the full dynamic range of 0 - 255 pixel values may
>
>
>
>  
> > likely be reproduced with no details at the bright end (240 - 255), and
>
>
>
>  
> > the dark end (0 - 20).  Printing presses cannot reproduce detail within
>
>
>
>  
> > these tonal ranges because of limitations with dropping ink on paper
>
>
>
>  
> > without A) having the drop not stick when the tonal values are bright
>
>
>
>  
> > and B) having the inks blend into each other through capillary action
>
>
>
>  
> > at the darkest values.  This phenomenon gets worse when the paper is
>
>
>
>  
> > lower quality (e.g., "Science").<br>
>
>
>
>  
>
>
>
>
>  
> So what we are saying is maybe summarised as follows:
>
>
>
>  
>
>
>
>
>  
> Small thumbnail images in print can only every be just that - pointers to go and
>
>
>
>  
> look at the real digital image on some online source.
>
>
>
>  
> No one can hope to reach quantitative conclusions from a small print image.
>
>
>
>  
>
>
>
>
>  
> We can probably consign print images on paper to the heap of old technologies,
>
>
>
>  
> which are no longer relevant to the work we do.
>
>
>
>  
>
>
>
>
>  
> Images in PDF files "could" be useful, but not if they are lossy compressed and
>
>
>
>  
> missing meta data, which they usually are.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > Color reproduction is generally worse because primary and secondary
>
>
>
>  
> > colors are used to show experimental evidence.  These colors are not
>
>
>
>  
> > always within the range of printing presses, and so these tend to print
>
>
>
>  
> > blobs sans detail when color choices are not appropriate for publishing
>
>
>
>  
> > onto paper.<br>
>
>
>
>  
>
>
>
>
>  
> To make matters worse....
>
>
>
>  
> fors most peoples eyes, blue is much fainter than red and green, with green
>
>
>
>  
> often being precieved as brightest.
>
>
>
>  
> So forget even semi  quantitative comparisons of intensity there.
>
>
>
>  
>
>
>
>
>  
> In any case we dont print in red green and blue, so these are silly choices for
>
>
>
>  
> a print version of figures.
>
>
>
>  
> The print colour space would be better, Magenta, yellow and cyan.... but still
>
>
>
>  
> for our quantitative purposed not good.
>
>
>
>  
>
>
>
>
>  
> Screen (LCD/CRT) colours are more similar to out eyes trichromatic response.
>
>
>
>  
> That why they are red green and blue... but the blue looks darker then green
>
>
>
>  
> problem still persists.
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > More often than not, the issue of whether or not details can be
>
>
>
>  
> > resolved by eye on a printed page is not that of how many pixels exist
>
>
>
>  
> > in the submitted image, but in how effectively tones and colors were
>
>
>
>  
> > fitted to the printing press.  This is especially true if the pdf image
>
>
>
>  
> > at non-zoomed, computer screen resolution reveals desired details, but
>
>
>
>  
> > the printed page does not:  it is more likely that a 133 line per inch
>
>
>
>  
> > screened image appearing in publication has more resolution than the
>
>
>
>  
> > computer screen (often figured at an average of 90 pixels per inch,
>
>
>
>  
> > with 72 pixels being the "old" standard).  The color and tonal range of
>
>
>
>  
> > reproduction of a computer screen is greater than on a printing press.<br>
>
>
>
>  
> > <br>
>
>
>
>  
>
>
>
>
>  
> Indeed it is, and so is a better visualisation tool...
>
>
>
>  
> but to get the most out of it, we also need to be smart.
>
>
>
>  
> We cant compare the brightness of blue and green due to our physiology,
>
>
>
>  
> even if the screen is properly calibrated (usually not the case).
>
>
>
>  
>
>
>
>
>  
> So, we should not ry to do that on screen or in print.
>
>
>
>  
>
>
>
>
>  
> Our eyes are much better at discriminating greyscale brightness scale,
>
>
>
>  
> but actually we are sill pretty bad at that.
>
>
>
>  
>
>
>
>
>  
> see this example of where we are easily fooled when trying to compare grey scale
>
>
>
>  
> brightness.
>
>
>
>  
> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>
>
>
>  
>
>
>
>
>  
> The situation with colouyr is more complicated, and also beset with pitfalle
>
>
>
>  
> due to the way our optical system works... it tries to find contrast in any
>
>
>
>  
> scene,
>
>
>
>  
> and even generates contrast or colour differences that are not there (but gave
>
>
>
>  
> us an evolutionary advantage in picking fruit)
>
>
>
>  
>
>
>
>
>  
> See the spiral image at the bottom f this colocalisation tutorial,
>
>
>
>  
> which shows us that we should be careful when interpreting colour merge multi
>
>
>
>  
> channel images.
>
>
>
>  
>
>
>
>
>  
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>
>
>  
>
>
>
>
>  
> you can draw the spiral pattern yourself in imageJ with the marco script there,
>
>
>
>  
> and prove to yourself that your eyes lie to you.
>
>
>
>  
>
>
>
>
>  
> Greyscale images can be made interpretable in a semi quantitative manner by
>
>
>
>  
> using for instance the fire colour look up table.
>
>
>
>  
> Each bright colour represents some intensity value, so its easier to compare
>
>
>
>  
> intensities over and between images.
>
>
>
>  
> Physicists and chemists do this all the time, and think we are mad for showing
>
>
>
>  
> DAPI staining  in black to Blue - because "thats what it looks like"
>
>
>
>  
> They are right , we are mad to do that.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > Like others before this email, I believe that it is best that
>
>
>
>  
> > scientists take the task of reproduction as much in their own hands as
>
>
>
>  
> > possible so that the outcome can be controlled.  The image is a
>
>
>
>  
> > reproduction of what was once under a microscope, and it behooves the
>
>
>
>  
> > researcher to make that appear as close to the original representation
>
>
>
>  
> > as possible.  <br>
>
>
>
>  
>
>
>
>
>  
> I'm not sure that this is ever going to be a helpful way to approach the
>
>
>
>  
> problem.
>
>
>
>  
> No matter how carefully you set out your images and send them to the publisher,
>
>
>
>  
> you have no control and what they do with them next. None.
>
>
>
>  
>
>
>
>
>  
> The only way around this is to have the images in the paper act only as
>
>
>
>  
> thumbnails
>
>
>
>  
> which point to the original image data file(s) on an online repository,
>
>
>
>  
> like the JCB image viewer or our wished for public biological image database...
>
>
>
>  
>
>
>
>
>  
> An image is not the sample, it always contain much less info that the sample
>
>
>
>  
> did.
>
>
>
>  
> The trick is to keep the useful info as it passes through the scope lenses,
>
>
>
>  
> the detector, and the computer you your brain/eyes.
>
>
>
>  
> I agree, an image is a representation of the info that made it through the
>
>
>
>  
> microscope to you.
>
>
>
>  
> Trying to represent it "faithfully" and as  it "truly looks" are both missing
>
>
>
>  
> the mark.
>
>
>
>  
> The image is usually degraded by the blur of the Point spread function / OTF  
>
>
>
>  
> and by various sources of noise.
>
>
>
>  
> Thus, the image is an artifact in of itself. What we want to know about is the
>
>
>
>  
> sample.
>
>
>
>  
> The images contains info from the sample in a degraded  and incomplete from.
>
>
>
>  
> We have to work around that, and not pretend that the image fully represents the
>
>
>
>  
> sample.
>
>
>
>  
> It does not.
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > As far as images being data points, this also is true, and these are to
>
>
>
>  
> > remain unaltered (unless flatfield correcting, background subtracting,
>
>
>
>  
> > etc) for measuring.  A faithful representation of that image when
>
>
>
>  
> > reproduced is another matter altogether.<br>
>
>
>
>  
>
>
>
>
>  
> Indeed it is, and i think there is even no such thing as a faithful
>
>
>
>  
> representation of the Sample as an image,
>
>
>
>  
> so the faithful representation of the image is also then something to think
>
>
>
>  
> about.
>
>
>
>  
>
>
>
>
>  
> I think what matters is How you represent the image data....
>
>
>
>  
> in some cases an illustration like Hooke's drawing of cells might even be better
>
>
>
>  
> than a digital representation,
>
>
>
>  
> if you want to get a certain message across to the reader. Original digital
>
>
>
>  
> image data available too of course online.  
>
>
>
>  
>
>
>
>
>  
> cheers
>
>
>
>  
>
>
>
>
>  
> Dan
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
>  
>
>
>
>  
> > <br>
>
>
>
>  
> > Cheers,<br>
>
>
>
>  
> > <br>
>
>
>
>  
> > Jerry Sedgewick<br>
>
>
>
>  
>
>
>
>
>  
> Dr. Daniel James White BSc. (Hons.) PhD
>
>
>
>  
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
>
>  
> Light Microscopy and Image Processing Facilities
>
>
>
>  
> Max Planck Institute of Molecular Cell Biology and Genetics
>
>
>
>  
> Pfotenhauerstrasse 108
>
>
>
>  
> 01307 DRESDEN
>
>
>
>  
> Germany
>
>
>
>  
>
>
>
>
>  
> [phone] (German Mobile)
>
>
>
>  
> [phone] (Work phone at MPI-CBG)
>
>
>
>  
> [phone] (Fax MPI-CBG LMF)
>
>
>
>  
>
>
>
>
>  
> http://www.bioimagexd.net   BioImageXD
>
>
>
>  
> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries Included)
>
>
>
>  
> http://www.chalkie.org.uk       Dan's Homepages
>
>
>
>  
> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>
>
>
>  
> dan (at) chalkie.org.uk
>
>
>
>  
> ( white (at) mpi-cbg.de )
>
>
>
>  
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
> ---------------------------------------------------------------------

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

David,

              I suppose it's partly Photoshop trying to guess what users
want, and partly a matter of what's computationally possible.  If you
are talking about 8-bit indexed color (ie grayscale with a palette
added) there is no way you can you can apply any of the sophisticated
resampling algorithms to it.  Turning it into RGB and resampling is
indeed wrong, very wrong, what you must do is turn it into grayscale and
resample, then re-apply the color palette.  The best resampling
algorithm is bicubic interpolation (see my chapter in the Pawley book,
where I compare algorithms).  Note that when converting the image to
grayscale you must do it by applying a grayscale palette while
maintaining indices, not using  'closest match' which will give you
nonsense.  

              Generally I don't use Photoshop, I use Paint Shop Pro, but
the basic steps should be similar.  Generate a gray-scale image with all
256 values present, and save the palette as a palette file (I call it
lin_grey).  Open the user's image and save the palette as another
palette file.  Now replace the palette, maintaining indices, by
lin_grey.  You now have a grayscale image which you can scale
effectively.  Scale with bicubic interpolation to the required value,
then re-apply the original palette (again, of course, maintaining
indices).  

                Is this kosher? Absolutely, 100%.  Your image IS a
gray-scale image, the palette is just an add-on.  It's no different from
enlarging a negative in a darkroom enlarger.  In the end your picture
will be represented in the printed page by an array of dots, between 120
and 300 to the inch, depending on the quality of the journal.  Your goal
is to get the data to convert accurately to this representation.

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of David Knecht
Sent: Thursday, 8 September 2011 8:25 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have been forced back into this issue in helping someone get an image
taken on our microscopes through a journal editor.  The images were
fairly low resolution.  They were manipulated in Photoshop, imported
into Canvas, text added and then output for the journal as a figure at
300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
the images were indexed color, the up sampling was done by taking each
pixel and subdividing it into smaller pixels.  Thus the pixelation of
the original was maintained and the editor did not like that.  What they
wanted done was to convert the images to RGB and then upsample.  In that
case (or grayscale), Photoshop does an interpolation making a smoother
looking zoomed in view, but that is a change in the data.  The index
version is actually more accurate although less pleasing. I plan to
argue that this is not necessary or desirable, but I did not know that
Photoshop (but not ImageJ) makes this distinction and thought others
might want to know it happens.  Does anyone know why index vs. RGB
should matter to the up sampling algorithm.  THanks- Dave


On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:

> Well, as George McNamara has suggested many times on the list the
image you should send in the "mean image", (an example of the images
collected).
> I would predict that this is rarely the case and that most images sent
to journals are extreme examples of the best possible image collected
and corrected.
> However, sending the entire image set (in Gigabytes or even Terabytes)
to the journal seems impractical. Perhaps instead one could acquire the
entire data set by e-mailing the author.  

>  
>  
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
> From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Andreas
Bruckbauer
> Sent: Tuesday, July 13, 2010 2:14 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>  
> Dear all,
> so far this discussion mainly focusses on "the  image" but one image
alone can not represent the data. Think about the data set  represented
by a distribution  which might be gaussian, so you would  need  at
least the mean and  the  width of the  distribution. What i want to say
is that you will need to do a quantification and then show  an image
representing  the mean and show a histogram which gives the reader a
clue about how the data  is  distributed. There might be more than  one
variable which is  important for the  analysis and of  course you should
do an idependend repeat of the  experiment.
> So what would you suggest, sending all the original image files to the
data bank?
> I still think that rather than re-analysing other scientists data a
repeat of the  experiment in another lab is more important, there are a
lot of thinks apart  from data analysis and representation which can go
wrong. However i see the point that when the paper is about image
analysis to  provide the original files to let other groups  repreat the
analysis.  

>
> best wishes
>
> Andreas
>
>  
>  
>  
> -----Original Message-----
> From: Daniel James White <[hidden email]>
> To: [hidden email]
> Sent: Tue, 13 Jul 2010 9:01
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear Jerry,
>
>
>
>  
>
>
>
>
>  
> cheers for adding your valuable input to this discussion.
>
>
>
>  
> Its been a long and interesting one so far.
>
>
>
>  
>
>
>
>
>  
> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest

system wrote:

>
>
>
>  
>
>
>
>
>  
> > Date:    Mon, 12 Jul 2010 12:47:51 -0500
>
>
>
>  
> > From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>
>
>
>  
> > Subject: Re: Preparing figures for publication --PPI vs DPI
>
>
>
>  
> >
>
>
>
>  
> > <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>
>
>
>  
> > <html>
>
>
>
>  
> > <head>
>
>
>
>  
> >  <meta content="text/html; charset=windows-1252"
>
>
>
>  
> > http-equiv="Content-Type">
>
>
>
>  
> > </head>
>
>
>
>  
> > <body bgcolor="#ffffff" text="#000000">
>
>
>
>  
> > Hi All,<br>
>
>
>
>  
> > <br>
>
>
>
>  
>
>
>
>
>  
> maybe you might like to turn off HTML text formatting in your email

client when

>
>
>
>  
> you post to the lists
>
>
>
>  
> in this case its not so distracting but sometimes emails to this an

other list

>
>
>
>  
> as unreadable in digest mode
>
>
>
>  
> due to the thousands of html tags.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > I'm a little late in addressing this issue, but the PPI/DPI part of
>
>
>
>  
> > this conversation is misleading.  What I mean to say is that the

real

>
>
>
>  
> > issue may not be resolution, but reproduction.  
>
>
>
>  
>
>
>
>
>  
> A very good point.
>
>
>
>  
>
>
>
>
>  
> > Images that are sent to
>
>
>
>  
> > publishers with the full dynamic range of 0 - 255 pixel values may
>
>
>
>  
> > likely be reproduced with no details at the bright end (240 - 255),

and
>
>
>
>  
> > the dark end (0 - 20).  Printing presses cannot reproduce detail
within

>
>
>
>  
> > these tonal ranges because of limitations with dropping ink on paper
>
>
>
>  
> > without A) having the drop not stick when the tonal values are

bright
>
>
>
>  
> > and B) having the inks blend into each other through capillary
action

>
>
>
>  
> > at the darkest values.  This phenomenon gets worse when the paper is
>
>
>
>  
> > lower quality (e.g., "Science").<br>
>
>
>
>  
>
>
>
>
>  
> So what we are saying is maybe summarised as follows:
>
>
>
>  
>
>
>
>
>  
> Small thumbnail images in print can only every be just that - pointers

to go and

>
>
>
>  
> look at the real digital image on some online source.
>
>
>
>  
> No one can hope to reach quantitative conclusions from a small print

image.

>
>
>
>  
>
>
>
>
>  
> We can probably consign print images on paper to the heap of old

technologies,

>
>
>
>  
> which are no longer relevant to the work we do.
>
>
>
>  
>
>
>
>
>  
> Images in PDF files "could" be useful, but not if they are lossy

compressed and

>
>
>
>  
> missing meta data, which they usually are.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > Color reproduction is generally worse because primary and secondary
>
>
>
>  
> > colors are used to show experimental evidence.  These colors are not
>
>
>
>  
> > always within the range of printing presses, and so these tend to

print
>
>
>
>  
> > blobs sans detail when color choices are not appropriate for
publishing

>
>
>
>  
> > onto paper.<br>
>
>
>
>  
>
>
>
>
>  
> To make matters worse....
>
>
>
>  
> fors most peoples eyes, blue is much fainter than red and green, with

green

>
>
>
>  
> often being precieved as brightest.
>
>
>
>  
> So forget even semi  quantitative comparisons of intensity there.
>
>
>
>  
>
>
>
>
>  
> In any case we dont print in red green and blue, so these are silly

choices for

>
>
>
>  
> a print version of figures.
>
>
>
>  
> The print colour space would be better, Magenta, yellow and cyan....

but still

>
>
>
>  
> for our quantitative purposed not good.
>
>
>
>  
>
>
>
>
>  
> Screen (LCD/CRT) colours are more similar to out eyes trichromatic

response.
>
>
>
>  
> That why they are red green and blue... but the blue looks darker then
green

>
>
>
>  
> problem still persists.
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > More often than not, the issue of whether or not details can be
>
>
>
>  
> > resolved by eye on a printed page is not that of how many pixels

exist

>
>
>
>  
> > in the submitted image, but in how effectively tones and colors were
>
>
>
>  
> > fitted to the printing press.  This is especially true if the pdf

image
>
>
>
>  
> > at non-zoomed, computer screen resolution reveals desired details,
but
>
>
>
>  
> > the printed page does not:  it is more likely that a 133 line per
inch

>
>
>
>  
> > screened image appearing in publication has more resolution than the
>
>
>
>  
> > computer screen (often figured at an average of 90 pixels per inch,
>
>
>
>  
> > with 72 pixels being the "old" standard).  The color and tonal range

of
>
>
>
>  
> > reproduction of a computer screen is greater than on a printing
press.<br>

>
>
>
>  
> > <br>
>
>
>
>  
>
>
>
>
>  
> Indeed it is, and so is a better visualisation tool...
>
>
>
>  
> but to get the most out of it, we also need to be smart.
>
>
>
>  
> We cant compare the brightness of blue and green due to our

physiology,

>
>
>
>  
> even if the screen is properly calibrated (usually not the case).
>
>
>
>  
>
>
>
>
>  
> So, we should not ry to do that on screen or in print.
>
>
>
>  
>
>
>
>
>  
> Our eyes are much better at discriminating greyscale brightness scale,

>
>
>
>  
> but actually we are sill pretty bad at that.
>
>
>
>  
>
>
>
>
>  
> see this example of where we are easily fooled when trying to compare

grey scale

>
>
>
>  
> brightness.
>
>
>
>  
> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>
>
>
>  
>
>
>
>
>  
> The situation with colouyr is more complicated, and also beset with

pitfalle
>
>
>
>  
> due to the way our optical system works... it tries to find contrast
in any

>
>
>
>  
> scene,
>
>
>
>  
> and even generates contrast or colour differences that are not there

(but gave

>
>
>
>  
> us an evolutionary advantage in picking fruit)
>
>
>
>  
>
>
>
>
>  
> See the spiral image at the bottom f this colocalisation tutorial,
>
>
>
>  
> which shows us that we should be careful when interpreting colour

merge multi

>
>
>
>  
> channel images.
>
>
>
>  
>
>
>
>
>  
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>
>
>  
>
>
>
>
>  
> you can draw the spiral pattern yourself in imageJ with the marco

script there,

>
>
>
>  
> and prove to yourself that your eyes lie to you.
>
>
>
>  
>
>
>
>
>  
> Greyscale images can be made interpretable in a semi quantitative

manner by

>
>
>
>  
> using for instance the fire colour look up table.
>
>
>
>  
> Each bright colour represents some intensity value, so its easier to

compare

>
>
>
>  
> intensities over and between images.
>
>
>
>  
> Physicists and chemists do this all the time, and think we are mad for

showing

>
>
>
>  
> DAPI staining  in black to Blue - because "thats what it looks like"
>
>
>
>  
> They are right , we are mad to do that.
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
> > Like others before this email, I believe that it is best that
>
>
>
>  
> > scientists take the task of reproduction as much in their own hands

as

>
>
>
>  
> > possible so that the outcome can be controlled.  The image is a
>
>
>
>  
> > reproduction of what was once under a microscope, and it behooves

the
>
>
>
>  
> > researcher to make that appear as close to the original
representation

>
>
>
>  
> > as possible.  <br>
>
>
>
>  
>
>
>
>
>  
> I'm not sure that this is ever going to be a helpful way to approach

the

>
>
>
>  
> problem.
>
>
>
>  
> No matter how carefully you set out your images and send them to the

publisher,

>
>
>
>  
> you have no control and what they do with them next. None.
>
>
>
>  
>
>
>
>
>  
> The only way around this is to have the images in the paper act only

as

>
>
>
>  
> thumbnails
>
>
>
>  
> which point to the original image data file(s) on an online

repository,
>
>
>
>  
> like the JCB image viewer or our wished for public biological image
database...

>
>
>
>  
>
>
>
>
>  
> An image is not the sample, it always contain much less info that the

sample

>
>
>
>  
> did.
>
>
>
>  
> The trick is to keep the useful info as it passes through the scope

lenses,

>
>
>
>  
> the detector, and the computer you your brain/eyes.
>
>
>
>  
> I agree, an image is a representation of the info that made it through

the

>
>
>
>  
> microscope to you.
>
>
>
>  
> Trying to represent it "faithfully" and as  it "truly looks" are both

missing

>
>
>
>  
> the mark.
>
>
>
>  
> The image is usually degraded by the blur of the Point spread function

/ OTF  

>
>
>
>  
> and by various sources of noise.
>
>
>
>  
> Thus, the image is an artifact in of itself. What we want to know

about is the

>
>
>
>  
> sample.
>
>
>
>  
> The images contains info from the sample in a degraded  and incomplete

from.
>
>
>
>  
> We have to work around that, and not pretend that the image fully
represents the

>
>
>
>  
> sample.
>
>
>
>  
> It does not.
>
>
>
>  
>
>
>
>
>  
> > <br>
>
>
>
>  
> > As far as images being data points, this also is true, and these are

to
>
>
>
>  
> > remain unaltered (unless flatfield correcting, background
subtracting,

>
>
>
>  
> > etc) for measuring.  A faithful representation of that image when
>
>
>
>  
> > reproduced is another matter altogether.<br>
>
>
>
>  
>
>
>
>
>  
> Indeed it is, and i think there is even no such thing as a faithful
>
>
>
>  
> representation of the Sample as an image,
>
>
>
>  
> so the faithful representation of the image is also then something to

think

>
>
>
>  
> about.
>
>
>
>  
>
>
>
>
>  
> I think what matters is How you represent the image data....
>
>
>
>  
> in some cases an illustration like Hooke's drawing of cells might even

be better

>
>
>
>  
> than a digital representation,
>
>
>
>  
> if you want to get a certain message across to the reader. Original

digital

>
>
>
>  
> image data available too of course online.  
>
>
>
>  
>
>
>
>
>  
> cheers
>
>
>
>  
>
>
>
>
>  
> Dan
>
>
>
>  
>
>
>
>
>  
>
>
>
>
>  
>  
>
>
>
>  
> > <br>
>
>
>
>  
> > Cheers,<br>
>
>
>
>  
> > <br>
>
>
>
>  
> > Jerry Sedgewick<br>
>
>
>
>  
>
>
>
>
>  
> Dr. Daniel James White BSc. (Hons.) PhD
>
>
>
>  
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
>
>  
> Light Microscopy and Image Processing Facilities
>
>
>
>  
> Max Planck Institute of Molecular Cell Biology and Genetics
>
>
>
>  
> Pfotenhauerstrasse 108
>
>
>
>  
> 01307 DRESDEN
>
>
>
>  
> Germany
>
>
>
>  
>
>
>
>
>  
> [phone] (German Mobile)
>
>
>
>  
> [phone] (Work phone at MPI-CBG)
>
>
>
>  
> [phone] (Fax MPI-CBG LMF)
>
>
>
>  
>
>
>
>
>  
> http://www.bioimagexd.net   BioImageXD
>
>
>
>  
> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries

Included)

>
>
>
>  
> http://www.chalkie.org.uk       Dan's Homepages
>
>
>
>  
> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>
>
>
>  
> dan (at) chalkie.org.uk
>
>
>
>  
> ( white (at) mpi-cbg.de )
>
>
>
>  
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the

individual or entity to which they are addressed. This communication may
contain information that is privileged, confidential, or exempt from
disclosure under applicable law (e.g., personal health information,
research data, financial information). Because this e-mail has been sent
without encryption, individuals other than the intended recipient may be
able to view the information, forward it to others or tamper with the
information without the knowledge or consent of the sender. If you are
not the intended recipient, or the employee or person responsible for
delivering the message to the intended recipient, any dissemination,
distribution or copying of the communication is strictly prohibited. If
you received the communication in error, please notify the sender
immediately by replying to this message and deleting the message and any
accompanying files from your system. If, due to the security risks, you
do not wish to receive further communications via e-mail, please reply
to this message and inform the sender that you do not wish to receive
further e-mail from the sender.
> ---------------------------------------------------------------------

Dr. David Knecht    
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Armstrong, Brian]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Is the request to upload an image that has increased Dpi?
For this I would open the image in Photoshop, open image size dialogue box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
It seems to me that resampling a scientific image using an algorithm such as bicubic is interpolating pixels and therefore creating new data where it did not exist before.
Am I missing something?

Brian Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
1500 East Duarte Road
Duarte, CA 91010
626-256-4673 x62872

Light Microscopy Core Facility

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, September 08, 2011 5:15 AM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

David,

              I suppose it's partly Photoshop trying to guess what users
want, and partly a matter of what's computationally possible.  If you
are talking about 8-bit indexed color (ie grayscale with a palette
added) there is no way you can you can apply any of the sophisticated
resampling algorithms to it.  Turning it into RGB and resampling is
indeed wrong, very wrong, what you must do is turn it into grayscale and
resample, then re-apply the color palette.  The best resampling
algorithm is bicubic interpolation (see my chapter in the Pawley book,
where I compare algorithms).  Note that when converting the image to
grayscale you must do it by applying a grayscale palette while
maintaining indices, not using  'closest match' which will give you
nonsense.

              Generally I don't use Photoshop, I use Paint Shop Pro, but
the basic steps should be similar.  Generate a gray-scale image with all
256 values present, and save the palette as a palette file (I call it
lin_grey).  Open the user's image and save the palette as another
palette file.  Now replace the palette, maintaining indices, by
lin_grey.  You now have a grayscale image which you can scale
effectively.  Scale with bicubic interpolation to the required value,
then re-apply the original palette (again, of course, maintaining
indices).

                Is this kosher? Absolutely, 100%.  Your image IS a
gray-scale image, the palette is just an add-on.  It's no different from
enlarging a negative in a darkroom enlarger.  In the end your picture
will be represented in the printed page by an array of dots, between 120
and 300 to the inch, depending on the quality of the journal.  Your goal
is to get the data to convert accurately to this representation.

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of David Knecht
Sent: Thursday, 8 September 2011 8:25 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have been forced back into this issue in helping someone get an image
taken on our microscopes through a journal editor.  The images were
fairly low resolution.  They were manipulated in Photoshop, imported
into Canvas, text added and then output for the journal as a figure at
300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
the images were indexed color, the up sampling was done by taking each
pixel and subdividing it into smaller pixels.  Thus the pixelation of
the original was maintained and the editor did not like that.  What they
wanted done was to convert the images to RGB and then upsample.  In that
case (or grayscale), Photoshop does an interpolation making a smoother
looking zoomed in view, but that is a change in the data.  The index
version is actually more accurate although less pleasing. I plan to
argue that this is not necessary or desirable, but I did not know that
Photoshop (but not ImageJ) makes this distinction and thought others
might want to know it happens.  Does anyone know why index vs. RGB
should matter to the up sampling algorithm.  THanks- Dave


On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:

> Well, as George McNamara has suggested many times on the list the
image you should send in the "mean image", (an example of the images
collected).
> I would predict that this is rarely the case and that most images sent
to journals are extreme examples of the best possible image collected
and corrected.
> However, sending the entire image set (in Gigabytes or even Terabytes)
to the journal seems impractical. Perhaps instead one could acquire the
entire data set by e-mailing the author.

>
>
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
> From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Andreas
Bruckbauer
> Sent: Tuesday, July 13, 2010 2:14 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear all,
> so far this discussion mainly focusses on "the  image" but one image
alone can not represent the data. Think about the data set  represented
by a distribution  which might be gaussian, so you would  need  at
least the mean and  the  width of the  distribution. What i want to say
is that you will need to do a quantification and then show  an image
representing  the mean and show a histogram which gives the reader a
clue about how the data  is  distributed. There might be more than  one
variable which is  important for the  analysis and of  course you should
do an idependend repeat of the  experiment.
> So what would you suggest, sending all the original image files to the
data bank?
> I still think that rather than re-analysing other scientists data a
repeat of the  experiment in another lab is more important, there are a
lot of thinks apart  from data analysis and representation which can go
wrong. However i see the point that when the paper is about image
analysis to  provide the original files to let other groups  repreat the
analysis.

>
> best wishes
>
> Andreas
>
>
>
>
> -----Original Message-----
> From: Daniel James White <[hidden email]>
> To: [hidden email]
> Sent: Tue, 13 Jul 2010 9:01
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear Jerry,
>
>
>
>
>
>
>
>
>
> cheers for adding your valuable input to this discussion.
>
>
>
>
> Its been a long and interesting one so far.
>
>
>
>
>
>
>
>
>
> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest

system wrote:

>
>
>
>
>
>
>
>
>
> > Date:    Mon, 12 Jul 2010 12:47:51 -0500
>
>
>
>
> > From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>
>
>
>
> > Subject: Re: Preparing figures for publication --PPI vs DPI
>
>
>
>
> >
>
>
>
>
> > <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>
>
>
>
> > <html>
>
>
>
>
> > <head>
>
>
>
>
> >  <meta content="text/html; charset=windows-1252"
>
>
>
>
> > http-equiv="Content-Type">
>
>
>
>
> > </head>
>
>
>
>
> > <body bgcolor="#ffffff" text="#000000">
>
>
>
>
> > Hi All,<br>
>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> maybe you might like to turn off HTML text formatting in your email

client when

>
>
>
>
> you post to the lists
>
>
>
>
> in this case its not so distracting but sometimes emails to this an

other list

>
>
>
>
> as unreadable in digest mode
>
>
>
>
> due to the thousands of html tags.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > I'm a little late in addressing this issue, but the PPI/DPI part of
>
>
>
>
> > this conversation is misleading.  What I mean to say is that the

real

>
>
>
>
> > issue may not be resolution, but reproduction.
>
>
>
>
>
>
>
>
>
> A very good point.
>
>
>
>
>
>
>
>
>
> > Images that are sent to
>
>
>
>
> > publishers with the full dynamic range of 0 - 255 pixel values may
>
>
>
>
> > likely be reproduced with no details at the bright end (240 - 255),

and
>
>
>
>
> > the dark end (0 - 20).  Printing presses cannot reproduce detail
within

>
>
>
>
> > these tonal ranges because of limitations with dropping ink on paper
>
>
>
>
> > without A) having the drop not stick when the tonal values are

bright
>
>
>
>
> > and B) having the inks blend into each other through capillary
action

>
>
>
>
> > at the darkest values.  This phenomenon gets worse when the paper is
>
>
>
>
> > lower quality (e.g., "Science").<br>
>
>
>
>
>
>
>
>
>
> So what we are saying is maybe summarised as follows:
>
>
>
>
>
>
>
>
>
> Small thumbnail images in print can only every be just that - pointers

to go and

>
>
>
>
> look at the real digital image on some online source.
>
>
>
>
> No one can hope to reach quantitative conclusions from a small print

image.

>
>
>
>
>
>
>
>
>
> We can probably consign print images on paper to the heap of old

technologies,

>
>
>
>
> which are no longer relevant to the work we do.
>
>
>
>
>
>
>
>
>
> Images in PDF files "could" be useful, but not if they are lossy

compressed and

>
>
>
>
> missing meta data, which they usually are.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Color reproduction is generally worse because primary and secondary
>
>
>
>
> > colors are used to show experimental evidence.  These colors are not
>
>
>
>
> > always within the range of printing presses, and so these tend to

print
>
>
>
>
> > blobs sans detail when color choices are not appropriate for
publishing

>
>
>
>
> > onto paper.<br>
>
>
>
>
>
>
>
>
>
> To make matters worse....
>
>
>
>
> fors most peoples eyes, blue is much fainter than red and green, with

green

>
>
>
>
> often being precieved as brightest.
>
>
>
>
> So forget even semi  quantitative comparisons of intensity there.
>
>
>
>
>
>
>
>
>
> In any case we dont print in red green and blue, so these are silly

choices for

>
>
>
>
> a print version of figures.
>
>
>
>
> The print colour space would be better, Magenta, yellow and cyan....

but still

>
>
>
>
> for our quantitative purposed not good.
>
>
>
>
>
>
>
>
>
> Screen (LCD/CRT) colours are more similar to out eyes trichromatic

response.
>
>
>
>
> That why they are red green and blue... but the blue looks darker then
green

>
>
>
>
> problem still persists.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > More often than not, the issue of whether or not details can be
>
>
>
>
> > resolved by eye on a printed page is not that of how many pixels

exist

>
>
>
>
> > in the submitted image, but in how effectively tones and colors were
>
>
>
>
> > fitted to the printing press.  This is especially true if the pdf

image
>
>
>
>
> > at non-zoomed, computer screen resolution reveals desired details,
but
>
>
>
>
> > the printed page does not:  it is more likely that a 133 line per
inch

>
>
>
>
> > screened image appearing in publication has more resolution than the
>
>
>
>
> > computer screen (often figured at an average of 90 pixels per inch,
>
>
>
>
> > with 72 pixels being the "old" standard).  The color and tonal range

of
>
>
>
>
> > reproduction of a computer screen is greater than on a printing
press.<br>

>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and so is a better visualisation tool...
>
>
>
>
> but to get the most out of it, we also need to be smart.
>
>
>
>
> We cant compare the brightness of blue and green due to our

physiology,

>
>
>
>
> even if the screen is properly calibrated (usually not the case).
>
>
>
>
>
>
>
>
>
> So, we should not ry to do that on screen or in print.
>
>
>
>
>
>
>
>
>
> Our eyes are much better at discriminating greyscale brightness scale,

>
>
>
>
> but actually we are sill pretty bad at that.
>
>
>
>
>
>
>
>
>
> see this example of where we are easily fooled when trying to compare

grey scale

>
>
>
>
> brightness.
>
>
>
>
> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>
>
>
>
>
>
>
>
>
> The situation with colouyr is more complicated, and also beset with

pitfalle
>
>
>
>
> due to the way our optical system works... it tries to find contrast
in any

>
>
>
>
> scene,
>
>
>
>
> and even generates contrast or colour differences that are not there

(but gave

>
>
>
>
> us an evolutionary advantage in picking fruit)
>
>
>
>
>
>
>
>
>
> See the spiral image at the bottom f this colocalisation tutorial,
>
>
>
>
> which shows us that we should be careful when interpreting colour

merge multi

>
>
>
>
> channel images.
>
>
>
>
>
>
>
>
>
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>
>
>
>
>
>
>
>
> you can draw the spiral pattern yourself in imageJ with the marco

script there,

>
>
>
>
> and prove to yourself that your eyes lie to you.
>
>
>
>
>
>
>
>
>
> Greyscale images can be made interpretable in a semi quantitative

manner by

>
>
>
>
> using for instance the fire colour look up table.
>
>
>
>
> Each bright colour represents some intensity value, so its easier to

compare

>
>
>
>
> intensities over and between images.
>
>
>
>
> Physicists and chemists do this all the time, and think we are mad for

showing

>
>
>
>
> DAPI staining  in black to Blue - because "thats what it looks like"
>
>
>
>
> They are right , we are mad to do that.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > Like others before this email, I believe that it is best that
>
>
>
>
> > scientists take the task of reproduction as much in their own hands

as

>
>
>
>
> > possible so that the outcome can be controlled.  The image is a
>
>
>
>
> > reproduction of what was once under a microscope, and it behooves

the
>
>
>
>
> > researcher to make that appear as close to the original
representation

>
>
>
>
> > as possible.  <br>
>
>
>
>
>
>
>
>
>
> I'm not sure that this is ever going to be a helpful way to approach

the

>
>
>
>
> problem.
>
>
>
>
> No matter how carefully you set out your images and send them to the

publisher,

>
>
>
>
> you have no control and what they do with them next. None.
>
>
>
>
>
>
>
>
>
> The only way around this is to have the images in the paper act only

as

>
>
>
>
> thumbnails
>
>
>
>
> which point to the original image data file(s) on an online

repository,
>
>
>
>
> like the JCB image viewer or our wished for public biological image
database...

>
>
>
>
>
>
>
>
>
> An image is not the sample, it always contain much less info that the

sample

>
>
>
>
> did.
>
>
>
>
> The trick is to keep the useful info as it passes through the scope

lenses,

>
>
>
>
> the detector, and the computer you your brain/eyes.
>
>
>
>
> I agree, an image is a representation of the info that made it through

the

>
>
>
>
> microscope to you.
>
>
>
>
> Trying to represent it "faithfully" and as  it "truly looks" are both

missing

>
>
>
>
> the mark.
>
>
>
>
> The image is usually degraded by the blur of the Point spread function

/ OTF

>
>
>
>
> and by various sources of noise.
>
>
>
>
> Thus, the image is an artifact in of itself. What we want to know

about is the

>
>
>
>
> sample.
>
>
>
>
> The images contains info from the sample in a degraded  and incomplete

from.
>
>
>
>
> We have to work around that, and not pretend that the image fully
represents the

>
>
>
>
> sample.
>
>
>
>
> It does not.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > As far as images being data points, this also is true, and these are

to
>
>
>
>
> > remain unaltered (unless flatfield correcting, background
subtracting,

>
>
>
>
> > etc) for measuring.  A faithful representation of that image when
>
>
>
>
> > reproduced is another matter altogether.<br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and i think there is even no such thing as a faithful
>
>
>
>
> representation of the Sample as an image,
>
>
>
>
> so the faithful representation of the image is also then something to

think

>
>
>
>
> about.
>
>
>
>
>
>
>
>
>
> I think what matters is How you represent the image data....
>
>
>
>
> in some cases an illustration like Hooke's drawing of cells might even

be better

>
>
>
>
> than a digital representation,
>
>
>
>
> if you want to get a certain message across to the reader. Original

digital

>
>
>
>
> image data available too of course online.
>
>
>
>
>
>
>
>
>
> cheers
>
>
>
>
>
>
>
>
>
> Dan
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Cheers,<br>
>
>
>
>
> > <br>
>
>
>
>
> > Jerry Sedgewick<br>
>
>
>
>
>
>
>
>
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
>
>
>
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
>
>
> Light Microscopy and Image Processing Facilities
>
>
>
>
> Max Planck Institute of Molecular Cell Biology and Genetics
>
>
>
>
> Pfotenhauerstrasse 108
>
>
>
>
> 01307 DRESDEN
>
>
>
>
> Germany
>
>
>
>
>
>
>
>
>
> [phone] (German Mobile)
>
>
>
>
> [phone] (Work phone at MPI-CBG)
>
>
>
>
> [phone] (Fax MPI-CBG LMF)
>
>
>
>
>
>
>
>
>
> http://www.bioimagexd.net   BioImageXD
>
>
>
>
> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries

Included)

>
>
>
>
> http://www.chalkie.org.uk       Dan's Homepages
>
>
>
>
> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>
>
>
>
> dan (at) chalkie.org.uk
>
>
>
>
> ( white (at) mpi-cbg.de )
>
>
>
>
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the

individual or entity to which they are addressed. This communication may
contain information that is privileged, confidential, or exempt from
disclosure under applicable law (e.g., personal health information,
research data, financial information). Because this e-mail has been sent
without encryption, individuals other than the intended recipient may be
able to view the information, forward it to others or tamper with the
information without the knowledge or consent of the sender. If you are
not the intended recipient, or the employee or person responsible for
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you received the communication in error, please notify the sender
immediately by replying to this message and deleting the message and any
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do not wish to receive further communications via e-mail, please reply
to this message and inform the sender that you do not wish to receive
further e-mail from the sender.
> ---------------------------------------------------------------------

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[mcammer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Part of the problem is that instructions given by the journals are inconsistent.

They tell us to not manipulate the resolution, but then they insist that images be submitted in PDF format with JPG compression or that they be resized up to 300 to 600 DPI to look smooth.  And then everything is arbitrarily downsized when put on the web and who knows what interpolations are done for the print versions.

But practically, is this really a problem?  We used to use film and chemicals to process which were notorious for having variable responses.  Why wasn't image manipulation so controversial back when we made figures by rephotographing physically collaged photographs and then intentionally making the backgrounds gray in our darkroom processed prints because reprinting in journals lost the bottom 10% or so of grays in the black ink.  And the journals rephotographed the figures and halftoned them before the printing presses inconsistently inked the plates and squished onto variably coated papers.

It wasn't a problem because we were illustrating our manuscripts, just as we do now.  And, in fact, we have more control now with linear large dynamic range detectors and control of each discrete voxel up until the very last moment of spatial rescaling for publication.  Relative intensities remain invariable through every step of figure production, or if one condition is changed, such as gamma, all (control and experimentals) are changed according to the same method and we can describe the precise math used.  And often these changes are made to explain difficult to see details.

How about on the date of publication all imaging raw data be released on the web and linked to the publication?  Anyone who doesn't trust the illustrations could look at the raw data.  This is practical.

________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: Thursday, September 08, 2011 1:32 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Is the request to upload an image that has increased Dpi?
For this I would open the image in Photoshop, open image size dialogue box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
It seems to me that resampling a scientific image using an algorithm such as bicubic is interpolating pixels and therefore creating new data where it did not exist before.
Am I missing something?

Brian Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
1500 East Duarte Road
Duarte, CA 91010
626-256-4673 x62872

Light Microscopy Core Facility

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, September 08, 2011 5:15 AM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

David,

              I suppose it's partly Photoshop trying to guess what users
want, and partly a matter of what's computationally possible.  If you
are talking about 8-bit indexed color (ie grayscale with a palette
added) there is no way you can you can apply any of the sophisticated
resampling algorithms to it.  Turning it into RGB and resampling is
indeed wrong, very wrong, what you must do is turn it into grayscale and
resample, then re-apply the color palette.  The best resampling
algorithm is bicubic interpolation (see my chapter in the Pawley book,
where I compare algorithms).  Note that when converting the image to
grayscale you must do it by applying a grayscale palette while
maintaining indices, not using  'closest match' which will give you
nonsense.

              Generally I don't use Photoshop, I use Paint Shop Pro, but
the basic steps should be similar.  Generate a gray-scale image with all
256 values present, and save the palette as a palette file (I call it
lin_grey).  Open the user's image and save the palette as another
palette file.  Now replace the palette, maintaining indices, by
lin_grey.  You now have a grayscale image which you can scale
effectively.  Scale with bicubic interpolation to the required value,
then re-apply the original palette (again, of course, maintaining
indices).

                Is this kosher? Absolutely, 100%.  Your image IS a
gray-scale image, the palette is just an add-on.  It's no different from
enlarging a negative in a darkroom enlarger.  In the end your picture
will be represented in the printed page by an array of dots, between 120
and 300 to the inch, depending on the quality of the journal.  Your goal
is to get the data to convert accurately to this representation.

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of David Knecht
Sent: Thursday, 8 September 2011 8:25 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have been forced back into this issue in helping someone get an image
taken on our microscopes through a journal editor.  The images were
fairly low resolution.  They were manipulated in Photoshop, imported
into Canvas, text added and then output for the journal as a figure at
300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
the images were indexed color, the up sampling was done by taking each
pixel and subdividing it into smaller pixels.  Thus the pixelation of
the original was maintained and the editor did not like that.  What they
wanted done was to convert the images to RGB and then upsample.  In that
case (or grayscale), Photoshop does an interpolation making a smoother
looking zoomed in view, but that is a change in the data.  The index
version is actually more accurate although less pleasing. I plan to
argue that this is not necessary or desirable, but I did not know that
Photoshop (but not ImageJ) makes this distinction and thought others
might want to know it happens.  Does anyone know why index vs. RGB
should matter to the up sampling algorithm.  THanks- Dave


On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:

> Well, as George McNamara has suggested many times on the list the
image you should send in the "mean image", (an example of the images
collected).
> I would predict that this is rarely the case and that most images sent
to journals are extreme examples of the best possible image collected
and corrected.
> However, sending the entire image set (in Gigabytes or even Terabytes)
to the journal seems impractical. Perhaps instead one could acquire the
entire data set by e-mailing the author.

>
>
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
> From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Andreas
Bruckbauer
> Sent: Tuesday, July 13, 2010 2:14 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear all,
> so far this discussion mainly focusses on "the  image" but one image
alone can not represent the data. Think about the data set  represented
by a distribution  which might be gaussian, so you would  need  at
least the mean and  the  width of the  distribution. What i want to say
is that you will need to do a quantification and then show  an image
representing  the mean and show a histogram which gives the reader a
clue about how the data  is  distributed. There might be more than  one
variable which is  important for the  analysis and of  course you should
do an idependend repeat of the  experiment.
> So what would you suggest, sending all the original image files to the
data bank?
> I still think that rather than re-analysing other scientists data a
repeat of the  experiment in another lab is more important, there are a
lot of thinks apart  from data analysis and representation which can go
wrong. However i see the point that when the paper is about image
analysis to  provide the original files to let other groups  repreat the
analysis.

>
> best wishes
>
> Andreas
>
>
>
>
> -----Original Message-----
> From: Daniel James White <[hidden email]>
> To: [hidden email]
> Sent: Tue, 13 Jul 2010 9:01
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear Jerry,
>
>
>
>
>
>
>
>
>
> cheers for adding your valuable input to this discussion.
>
>
>
>
> Its been a long and interesting one so far.
>
>
>
>
>
>
>
>
>
> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest

system wrote:

>
>
>
>
>
>
>
>
>
> > Date:    Mon, 12 Jul 2010 12:47:51 -0500
>
>
>
>
> > From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>
>
>
>
> > Subject: Re: Preparing figures for publication --PPI vs DPI
>
>
>
>
> >
>
>
>
>
> > <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>
>
>
>
> > <html>
>
>
>
>
> > <head>
>
>
>
>
> >  <meta content="text/html; charset=windows-1252"
>
>
>
>
> > http-equiv="Content-Type">
>
>
>
>
> > </head>
>
>
>
>
> > <body bgcolor="#ffffff" text="#000000">
>
>
>
>
> > Hi All,<br>
>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> maybe you might like to turn off HTML text formatting in your email

client when

>
>
>
>
> you post to the lists
>
>
>
>
> in this case its not so distracting but sometimes emails to this an

other list

>
>
>
>
> as unreadable in digest mode
>
>
>
>
> due to the thousands of html tags.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > I'm a little late in addressing this issue, but the PPI/DPI part of
>
>
>
>
> > this conversation is misleading.  What I mean to say is that the

real

>
>
>
>
> > issue may not be resolution, but reproduction.
>
>
>
>
>
>
>
>
>
> A very good point.
>
>
>
>
>
>
>
>
>
> > Images that are sent to
>
>
>
>
> > publishers with the full dynamic range of 0 - 255 pixel values may
>
>
>
>
> > likely be reproduced with no details at the bright end (240 - 255),

and
>
>
>
>
> > the dark end (0 - 20).  Printing presses cannot reproduce detail
within

>
>
>
>
> > these tonal ranges because of limitations with dropping ink on paper
>
>
>
>
> > without A) having the drop not stick when the tonal values are

bright
>
>
>
>
> > and B) having the inks blend into each other through capillary
action

>
>
>
>
> > at the darkest values.  This phenomenon gets worse when the paper is
>
>
>
>
> > lower quality (e.g., "Science").<br>
>
>
>
>
>
>
>
>
>
> So what we are saying is maybe summarised as follows:
>
>
>
>
>
>
>
>
>
> Small thumbnail images in print can only every be just that - pointers

to go and

>
>
>
>
> look at the real digital image on some online source.
>
>
>
>
> No one can hope to reach quantitative conclusions from a small print

image.

>
>
>
>
>
>
>
>
>
> We can probably consign print images on paper to the heap of old

technologies,

>
>
>
>
> which are no longer relevant to the work we do.
>
>
>
>
>
>
>
>
>
> Images in PDF files "could" be useful, but not if they are lossy

compressed and

>
>
>
>
> missing meta data, which they usually are.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Color reproduction is generally worse because primary and secondary
>
>
>
>
> > colors are used to show experimental evidence.  These colors are not
>
>
>
>
> > always within the range of printing presses, and so these tend to

print
>
>
>
>
> > blobs sans detail when color choices are not appropriate for
publishing

>
>
>
>
> > onto paper.<br>
>
>
>
>
>
>
>
>
>
> To make matters worse....
>
>
>
>
> fors most peoples eyes, blue is much fainter than red and green, with

green

>
>
>
>
> often being precieved as brightest.
>
>
>
>
> So forget even semi  quantitative comparisons of intensity there.
>
>
>
>
>
>
>
>
>
> In any case we dont print in red green and blue, so these are silly

choices for

>
>
>
>
> a print version of figures.
>
>
>
>
> The print colour space would be better, Magenta, yellow and cyan....

but still

>
>
>
>
> for our quantitative purposed not good.
>
>
>
>
>
>
>
>
>
> Screen (LCD/CRT) colours are more similar to out eyes trichromatic

response.
>
>
>
>
> That why they are red green and blue... but the blue looks darker then
green

>
>
>
>
> problem still persists.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > More often than not, the issue of whether or not details can be
>
>
>
>
> > resolved by eye on a printed page is not that of how many pixels

exist

>
>
>
>
> > in the submitted image, but in how effectively tones and colors were
>
>
>
>
> > fitted to the printing press.  This is especially true if the pdf

image
>
>
>
>
> > at non-zoomed, computer screen resolution reveals desired details,
but
>
>
>
>
> > the printed page does not:  it is more likely that a 133 line per
inch

>
>
>
>
> > screened image appearing in publication has more resolution than the
>
>
>
>
> > computer screen (often figured at an average of 90 pixels per inch,
>
>
>
>
> > with 72 pixels being the "old" standard).  The color and tonal range

of
>
>
>
>
> > reproduction of a computer screen is greater than on a printing
press.<br>

>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and so is a better visualisation tool...
>
>
>
>
> but to get the most out of it, we also need to be smart.
>
>
>
>
> We cant compare the brightness of blue and green due to our

physiology,

>
>
>
>
> even if the screen is properly calibrated (usually not the case).
>
>
>
>
>
>
>
>
>
> So, we should not ry to do that on screen or in print.
>
>
>
>
>
>
>
>
>
> Our eyes are much better at discriminating greyscale brightness scale,

>
>
>
>
> but actually we are sill pretty bad at that.
>
>
>
>
>
>
>
>
>
> see this example of where we are easily fooled when trying to compare

grey scale

>
>
>
>
> brightness.
>
>
>
>
> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>
>
>
>
>
>
>
>
>
> The situation with colouyr is more complicated, and also beset with

pitfalle
>
>
>
>
> due to the way our optical system works... it tries to find contrast
in any

>
>
>
>
> scene,
>
>
>
>
> and even generates contrast or colour differences that are not there

(but gave

>
>
>
>
> us an evolutionary advantage in picking fruit)
>
>
>
>
>
>
>
>
>
> See the spiral image at the bottom f this colocalisation tutorial,
>
>
>
>
> which shows us that we should be careful when interpreting colour

merge multi

>
>
>
>
> channel images.
>
>
>
>
>
>
>
>
>
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>
>
>
>
>
>
>
>
> you can draw the spiral pattern yourself in imageJ with the marco

script there,

>
>
>
>
> and prove to yourself that your eyes lie to you.
>
>
>
>
>
>
>
>
>
> Greyscale images can be made interpretable in a semi quantitative

manner by

>
>
>
>
> using for instance the fire colour look up table.
>
>
>
>
> Each bright colour represents some intensity value, so its easier to

compare

>
>
>
>
> intensities over and between images.
>
>
>
>
> Physicists and chemists do this all the time, and think we are mad for

showing

>
>
>
>
> DAPI staining  in black to Blue - because "thats what it looks like"
>
>
>
>
> They are right , we are mad to do that.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > Like others before this email, I believe that it is best that
>
>
>
>
> > scientists take the task of reproduction as much in their own hands

as

>
>
>
>
> > possible so that the outcome can be controlled.  The image is a
>
>
>
>
> > reproduction of what was once under a microscope, and it behooves

the
>
>
>
>
> > researcher to make that appear as close to the original
representation

>
>
>
>
> > as possible.  <br>
>
>
>
>
>
>
>
>
>
> I'm not sure that this is ever going to be a helpful way to approach

the

>
>
>
>
> problem.
>
>
>
>
> No matter how carefully you set out your images and send them to the

publisher,

>
>
>
>
> you have no control and what they do with them next. None.
>
>
>
>
>
>
>
>
>
> The only way around this is to have the images in the paper act only

as

>
>
>
>
> thumbnails
>
>
>
>
> which point to the original image data file(s) on an online

repository,
>
>
>
>
> like the JCB image viewer or our wished for public biological image
database...

>
>
>
>
>
>
>
>
>
> An image is not the sample, it always contain much less info that the

sample

>
>
>
>
> did.
>
>
>
>
> The trick is to keep the useful info as it passes through the scope

lenses,

>
>
>
>
> the detector, and the computer you your brain/eyes.
>
>
>
>
> I agree, an image is a representation of the info that made it through

the

>
>
>
>
> microscope to you.
>
>
>
>
> Trying to represent it "faithfully" and as  it "truly looks" are both

missing

>
>
>
>
> the mark.
>
>
>
>
> The image is usually degraded by the blur of the Point spread function

/ OTF

>
>
>
>
> and by various sources of noise.
>
>
>
>
> Thus, the image is an artifact in of itself. What we want to know

about is the

>
>
>
>
> sample.
>
>
>
>
> The images contains info from the sample in a degraded  and incomplete

from.
>
>
>
>
> We have to work around that, and not pretend that the image fully
represents the

>
>
>
>
> sample.
>
>
>
>
> It does not.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > As far as images being data points, this also is true, and these are

to
>
>
>
>
> > remain unaltered (unless flatfield correcting, background
subtracting,

>
>
>
>
> > etc) for measuring.  A faithful representation of that image when
>
>
>
>
> > reproduced is another matter altogether.<br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and i think there is even no such thing as a faithful
>
>
>
>
> representation of the Sample as an image,
>
>
>
>
> so the faithful representation of the image is also then something to

think

>
>
>
>
> about.
>
>
>
>
>
>
>
>
>
> I think what matters is How you represent the image data....
>
>
>
>
> in some cases an illustration like Hooke's drawing of cells might even

be better

>
>
>
>
> than a digital representation,
>
>
>
>
> if you want to get a certain message across to the reader. Original

digital

>
>
>
>
> image data available too of course online.
>
>
>
>
>
>
>
>
>
> cheers
>
>
>
>
>
>
>
>
>
> Dan
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Cheers,<br>
>
>
>
>
> > <br>
>
>
>
>
> > Jerry Sedgewick<br>
>
>
>
>
>
>
>
>
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
>
>
>
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
>
>
> Light Microscopy and Image Processing Facilities
>
>
>
>
> Max Planck Institute of Molecular Cell Biology and Genetics
>
>
>
>
> Pfotenhauerstrasse 108
>
>
>
>
> 01307 DRESDEN
>
>
>
>
> Germany
>
>
>
>
>
>
>
>
>
> [phone] (German Mobile)
>
>
>
>
> [phone] (Work phone at MPI-CBG)
>
>
>
>
> [phone] (Fax MPI-CBG LMF)
>
>
>
>
>
>
>
>
>
> http://www.bioimagexd.net   BioImageXD
>
>
>
>
> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries

Included)

>
>
>
>
> http://www.chalkie.org.uk       Dan's Homepages
>
>
>
>
> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>
>
>
>
> dan (at) chalkie.org.uk
>
>
>
>
> ( white (at) mpi-cbg.de )
>
>
>
>
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the

individual or entity to which they are addressed. This communication may
contain information that is privileged, confidential, or exempt from
disclosure under applicable law (e.g., personal health information,
research data, financial information). Because this e-mail has been sent
without encryption, individuals other than the intended recipient may be
able to view the information, forward it to others or tamper with the
information without the knowledge or consent of the sender. If you are
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do not wish to receive further communications via e-mail, please reply
to this message and inform the sender that you do not wish to receive
further e-mail from the sender.
> ---------------------------------------------------------------------

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)
</PRE>
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This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.<br />
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<PRE>

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Brian,

         In my view you are indeed missing something.  Nyquist sampling is ~2.3 pixels per resel.  At 300dpi these pixels would be 85µm in size, so you couldn't see structures that are resolved in the image.  A 512x512 image would be 43 mm or 1.7" square.  

           The aim of publication is communication, so to communicate your results you need to present them in a way that they can be understood when published in a journal.  In any case, a confocal image is just a series of sample points - Nyquist theory assumes that these will be presented as sine waves, not blocks.  So mapping your samples into a higher-resolution space is in fact the correct thing to do.  By all means put your original data into an archive, and I wish more journals would provide this facility (though equally I can understand why they don't).  

          If you are my age (or even half my age) you would once have recorded your images on 35mm film.  Did you insist on publishing them at that size (24x36mm)?  Of course not, you printed the negatives at the required size.  That's changing the data a LOT more than bicubic resampling.  

                                       Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
Sent: Friday, 9 September 2011 3:32 AM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Is the request to upload an image that has increased Dpi?
For this I would open the image in Photoshop, open image size dialogue box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
It seems to me that resampling a scientific image using an algorithm such as bicubic is interpolating pixels and therefore creating new data where it did not exist before.
Am I missing something?

Brian Armstrong PhD
Assistant Research Professor
Light Microscopy Core
Beckman Research Institute
City of Hope
1500 East Duarte Road
Duarte, CA 91010
626-256-4673 x62872

Light Microscopy Core Facility

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, September 08, 2011 5:15 AM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

David,

              I suppose it's partly Photoshop trying to guess what users
want, and partly a matter of what's computationally possible.  If you
are talking about 8-bit indexed color (ie grayscale with a palette
added) there is no way you can you can apply any of the sophisticated
resampling algorithms to it.  Turning it into RGB and resampling is
indeed wrong, very wrong, what you must do is turn it into grayscale and
resample, then re-apply the color palette.  The best resampling
algorithm is bicubic interpolation (see my chapter in the Pawley book,
where I compare algorithms).  Note that when converting the image to
grayscale you must do it by applying a grayscale palette while
maintaining indices, not using  'closest match' which will give you
nonsense.

              Generally I don't use Photoshop, I use Paint Shop Pro, but
the basic steps should be similar.  Generate a gray-scale image with all
256 values present, and save the palette as a palette file (I call it
lin_grey).  Open the user's image and save the palette as another
palette file.  Now replace the palette, maintaining indices, by
lin_grey.  You now have a grayscale image which you can scale
effectively.  Scale with bicubic interpolation to the required value,
then re-apply the original palette (again, of course, maintaining
indices).

                Is this kosher? Absolutely, 100%.  Your image IS a
gray-scale image, the palette is just an add-on.  It's no different from
enlarging a negative in a darkroom enlarger.  In the end your picture
will be represented in the printed page by an array of dots, between 120
and 300 to the inch, depending on the quality of the journal.  Your goal
is to get the data to convert accurately to this representation.

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of David Knecht
Sent: Thursday, 8 September 2011 8:25 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have been forced back into this issue in helping someone get an image
taken on our microscopes through a journal editor.  The images were
fairly low resolution.  They were manipulated in Photoshop, imported
into Canvas, text added and then output for the journal as a figure at
300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
the images were indexed color, the up sampling was done by taking each
pixel and subdividing it into smaller pixels.  Thus the pixelation of
the original was maintained and the editor did not like that.  What they
wanted done was to convert the images to RGB and then upsample.  In that
case (or grayscale), Photoshop does an interpolation making a smoother
looking zoomed in view, but that is a change in the data.  The index
version is actually more accurate although less pleasing. I plan to
argue that this is not necessary or desirable, but I did not know that
Photoshop (but not ImageJ) makes this distinction and thought others
might want to know it happens.  Does anyone know why index vs. RGB
should matter to the up sampling algorithm.  THanks- Dave


On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:

> Well, as George McNamara has suggested many times on the list the
image you should send in the "mean image", (an example of the images
collected).
> I would predict that this is rarely the case and that most images sent
to journals are extreme examples of the best possible image collected
and corrected.
> However, sending the entire image set (in Gigabytes or even Terabytes)
to the journal seems impractical. Perhaps instead one could acquire the
entire data set by e-mailing the author.

>
>
> Brian D Armstrong PhD
> Light Microscopy Core Manager
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx
> From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Andreas
Bruckbauer
> Sent: Tuesday, July 13, 2010 2:14 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear all,
> so far this discussion mainly focusses on "the  image" but one image
alone can not represent the data. Think about the data set  represented
by a distribution  which might be gaussian, so you would  need  at
least the mean and  the  width of the  distribution. What i want to say
is that you will need to do a quantification and then show  an image
representing  the mean and show a histogram which gives the reader a
clue about how the data  is  distributed. There might be more than  one
variable which is  important for the  analysis and of  course you should
do an idependend repeat of the  experiment.
> So what would you suggest, sending all the original image files to the
data bank?
> I still think that rather than re-analysing other scientists data a
repeat of the  experiment in another lab is more important, there are a
lot of thinks apart  from data analysis and representation which can go
wrong. However i see the point that when the paper is about image
analysis to  provide the original files to let other groups  repreat the
analysis.

>
> best wishes
>
> Andreas
>
>
>
>
> -----Original Message-----
> From: Daniel James White <[hidden email]>
> To: [hidden email]
> Sent: Tue, 13 Jul 2010 9:01
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> Dear Jerry,
>
>
>
>
>
>
>
>
>
> cheers for adding your valuable input to this discussion.
>
>
>
>
> Its been a long and interesting one so far.
>
>
>
>
>
>
>
>
>
> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest

system wrote:

>
>
>
>
>
>
>
>
>
> > Date:    Mon, 12 Jul 2010 12:47:51 -0500
>
>
>
>
> > From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>
>
>
>
> > Subject: Re: Preparing figures for publication --PPI vs DPI
>
>
>
>
> >
>
>
>
>
> > <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>
>
>
>
> > <html>
>
>
>
>
> > <head>
>
>
>
>
> >  <meta content="text/html; charset=windows-1252"
>
>
>
>
> > http-equiv="Content-Type">
>
>
>
>
> > </head>
>
>
>
>
> > <body bgcolor="#ffffff" text="#000000">
>
>
>
>
> > Hi All,<br>
>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> maybe you might like to turn off HTML text formatting in your email

client when

>
>
>
>
> you post to the lists
>
>
>
>
> in this case its not so distracting but sometimes emails to this an

other list

>
>
>
>
> as unreadable in digest mode
>
>
>
>
> due to the thousands of html tags.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > I'm a little late in addressing this issue, but the PPI/DPI part of
>
>
>
>
> > this conversation is misleading.  What I mean to say is that the

real

>
>
>
>
> > issue may not be resolution, but reproduction.
>
>
>
>
>
>
>
>
>
> A very good point.
>
>
>
>
>
>
>
>
>
> > Images that are sent to
>
>
>
>
> > publishers with the full dynamic range of 0 - 255 pixel values may
>
>
>
>
> > likely be reproduced with no details at the bright end (240 - 255),

and
>
>
>
>
> > the dark end (0 - 20).  Printing presses cannot reproduce detail
within

>
>
>
>
> > these tonal ranges because of limitations with dropping ink on paper
>
>
>
>
> > without A) having the drop not stick when the tonal values are

bright
>
>
>
>
> > and B) having the inks blend into each other through capillary
action

>
>
>
>
> > at the darkest values.  This phenomenon gets worse when the paper is
>
>
>
>
> > lower quality (e.g., "Science").<br>
>
>
>
>
>
>
>
>
>
> So what we are saying is maybe summarised as follows:
>
>
>
>
>
>
>
>
>
> Small thumbnail images in print can only every be just that - pointers

to go and

>
>
>
>
> look at the real digital image on some online source.
>
>
>
>
> No one can hope to reach quantitative conclusions from a small print

image.

>
>
>
>
>
>
>
>
>
> We can probably consign print images on paper to the heap of old

technologies,

>
>
>
>
> which are no longer relevant to the work we do.
>
>
>
>
>
>
>
>
>
> Images in PDF files "could" be useful, but not if they are lossy

compressed and

>
>
>
>
> missing meta data, which they usually are.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Color reproduction is generally worse because primary and secondary
>
>
>
>
> > colors are used to show experimental evidence.  These colors are not
>
>
>
>
> > always within the range of printing presses, and so these tend to

print
>
>
>
>
> > blobs sans detail when color choices are not appropriate for
publishing

>
>
>
>
> > onto paper.<br>
>
>
>
>
>
>
>
>
>
> To make matters worse....
>
>
>
>
> fors most peoples eyes, blue is much fainter than red and green, with

green

>
>
>
>
> often being precieved as brightest.
>
>
>
>
> So forget even semi  quantitative comparisons of intensity there.
>
>
>
>
>
>
>
>
>
> In any case we dont print in red green and blue, so these are silly

choices for

>
>
>
>
> a print version of figures.
>
>
>
>
> The print colour space would be better, Magenta, yellow and cyan....

but still

>
>
>
>
> for our quantitative purposed not good.
>
>
>
>
>
>
>
>
>
> Screen (LCD/CRT) colours are more similar to out eyes trichromatic

response.
>
>
>
>
> That why they are red green and blue... but the blue looks darker then
green

>
>
>
>
> problem still persists.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > More often than not, the issue of whether or not details can be
>
>
>
>
> > resolved by eye on a printed page is not that of how many pixels

exist

>
>
>
>
> > in the submitted image, but in how effectively tones and colors were
>
>
>
>
> > fitted to the printing press.  This is especially true if the pdf

image
>
>
>
>
> > at non-zoomed, computer screen resolution reveals desired details,
but
>
>
>
>
> > the printed page does not:  it is more likely that a 133 line per
inch

>
>
>
>
> > screened image appearing in publication has more resolution than the
>
>
>
>
> > computer screen (often figured at an average of 90 pixels per inch,
>
>
>
>
> > with 72 pixels being the "old" standard).  The color and tonal range

of
>
>
>
>
> > reproduction of a computer screen is greater than on a printing
press.<br>

>
>
>
>
> > <br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and so is a better visualisation tool...
>
>
>
>
> but to get the most out of it, we also need to be smart.
>
>
>
>
> We cant compare the brightness of blue and green due to our

physiology,

>
>
>
>
> even if the screen is properly calibrated (usually not the case).
>
>
>
>
>
>
>
>
>
> So, we should not ry to do that on screen or in print.
>
>
>
>
>
>
>
>
>
> Our eyes are much better at discriminating greyscale brightness scale,

>
>
>
>
> but actually we are sill pretty bad at that.
>
>
>
>
>
>
>
>
>
> see this example of where we are easily fooled when trying to compare

grey scale

>
>
>
>
> brightness.
>
>
>
>
> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>
>
>
>
>
>
>
>
>
> The situation with colouyr is more complicated, and also beset with

pitfalle
>
>
>
>
> due to the way our optical system works... it tries to find contrast
in any

>
>
>
>
> scene,
>
>
>
>
> and even generates contrast or colour differences that are not there

(but gave

>
>
>
>
> us an evolutionary advantage in picking fruit)
>
>
>
>
>
>
>
>
>
> See the spiral image at the bottom f this colocalisation tutorial,
>
>
>
>
> which shows us that we should be careful when interpreting colour

merge multi

>
>
>
>
> channel images.
>
>
>
>
>
>
>
>
>
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>
>
>
>
>
>
>
>
> you can draw the spiral pattern yourself in imageJ with the marco

script there,

>
>
>
>
> and prove to yourself that your eyes lie to you.
>
>
>
>
>
>
>
>
>
> Greyscale images can be made interpretable in a semi quantitative

manner by

>
>
>
>
> using for instance the fire colour look up table.
>
>
>
>
> Each bright colour represents some intensity value, so its easier to

compare

>
>
>
>
> intensities over and between images.
>
>
>
>
> Physicists and chemists do this all the time, and think we are mad for

showing

>
>
>
>
> DAPI staining  in black to Blue - because "thats what it looks like"
>
>
>
>
> They are right , we are mad to do that.
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > Like others before this email, I believe that it is best that
>
>
>
>
> > scientists take the task of reproduction as much in their own hands

as

>
>
>
>
> > possible so that the outcome can be controlled.  The image is a
>
>
>
>
> > reproduction of what was once under a microscope, and it behooves

the
>
>
>
>
> > researcher to make that appear as close to the original
representation

>
>
>
>
> > as possible.  <br>
>
>
>
>
>
>
>
>
>
> I'm not sure that this is ever going to be a helpful way to approach

the

>
>
>
>
> problem.
>
>
>
>
> No matter how carefully you set out your images and send them to the

publisher,

>
>
>
>
> you have no control and what they do with them next. None.
>
>
>
>
>
>
>
>
>
> The only way around this is to have the images in the paper act only

as

>
>
>
>
> thumbnails
>
>
>
>
> which point to the original image data file(s) on an online

repository,
>
>
>
>
> like the JCB image viewer or our wished for public biological image
database...

>
>
>
>
>
>
>
>
>
> An image is not the sample, it always contain much less info that the

sample

>
>
>
>
> did.
>
>
>
>
> The trick is to keep the useful info as it passes through the scope

lenses,

>
>
>
>
> the detector, and the computer you your brain/eyes.
>
>
>
>
> I agree, an image is a representation of the info that made it through

the

>
>
>
>
> microscope to you.
>
>
>
>
> Trying to represent it "faithfully" and as  it "truly looks" are both

missing

>
>
>
>
> the mark.
>
>
>
>
> The image is usually degraded by the blur of the Point spread function

/ OTF

>
>
>
>
> and by various sources of noise.
>
>
>
>
> Thus, the image is an artifact in of itself. What we want to know

about is the

>
>
>
>
> sample.
>
>
>
>
> The images contains info from the sample in a degraded  and incomplete

from.
>
>
>
>
> We have to work around that, and not pretend that the image fully
represents the

>
>
>
>
> sample.
>
>
>
>
> It does not.
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > As far as images being data points, this also is true, and these are

to
>
>
>
>
> > remain unaltered (unless flatfield correcting, background
subtracting,

>
>
>
>
> > etc) for measuring.  A faithful representation of that image when
>
>
>
>
> > reproduced is another matter altogether.<br>
>
>
>
>
>
>
>
>
>
> Indeed it is, and i think there is even no such thing as a faithful
>
>
>
>
> representation of the Sample as an image,
>
>
>
>
> so the faithful representation of the image is also then something to

think

>
>
>
>
> about.
>
>
>
>
>
>
>
>
>
> I think what matters is How you represent the image data....
>
>
>
>
> in some cases an illustration like Hooke's drawing of cells might even

be better

>
>
>
>
> than a digital representation,
>
>
>
>
> if you want to get a certain message across to the reader. Original

digital

>
>
>
>
> image data available too of course online.
>
>
>
>
>
>
>
>
>
> cheers
>
>
>
>
>
>
>
>
>
> Dan
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> > <br>
>
>
>
>
> > Cheers,<br>
>
>
>
>
> > <br>
>
>
>
>
> > Jerry Sedgewick<br>
>
>
>
>
>
>
>
>
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
>
>
>
> Senior Microscopist / Image Visualisation, Processing and Analysis
>
>
>
>
> Light Microscopy and Image Processing Facilities
>
>
>
>
> Max Planck Institute of Molecular Cell Biology and Genetics
>
>
>
>
> Pfotenhauerstrasse 108
>
>
>
>
> 01307 DRESDEN
>
>
>
>
> Germany
>
>
>
>
>
>
>
>
>
> [phone] (German Mobile)
>
>
>
>
> [phone] (Work phone at MPI-CBG)
>
>
>
>
> [phone] (Fax MPI-CBG LMF)
>
>
>
>
>
>
>
>
>
> http://www.bioimagexd.net   BioImageXD
>
>
>
>
> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries

Included)

>
>
>
>
> http://www.chalkie.org.uk       Dan's Homepages
>
>
>
>
> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>
>
>
>
> dan (at) chalkie.org.uk
>
>
>
>
> ( white (at) mpi-cbg.de )
>
>
>
>
>
> ---------------------------------------------------------------------
> SECURITY/CONFIDENTIALITY WARNING:
> This message and any attachments are intended solely for the

individual or entity to which they are addressed. This communication may
contain information that is privileged, confidential, or exempt from
disclosure under applicable law (e.g., personal health information,
research data, financial information). Because this e-mail has been sent
without encryption, individuals other than the intended recipient may be
able to view the information, forward it to others or tamper with the
information without the knowledge or consent of the sender. If you are
not the intended recipient, or the employee or person responsible for
delivering the message to the intended recipient, any dissemination,
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you received the communication in error, please notify the sender
immediately by replying to this message and deleting the message and any
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do not wish to receive further communications via e-mail, please reply
to this message and inform the sender that you do not wish to receive
further e-mail from the sender.
> ---------------------------------------------------------------------

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Daniel James White]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Dave and Listers,

I grow more polarized on this topic....

On Sep 9, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system wrote:

>
> Date:    Thu, 8 Sep 2011 11:24:52 +0100
> From:    David Knecht <[hidden email]>
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I have been forced back into this issue in helping someone get an image =
> taken on our microscopes through a journal editor.  The images were =
> fairly low resolution.

THe data is the data... there no no sensible reason to try to make it "look nicer"

>  They were manipulated in Photoshop, imported =
> into Canvas, text added and then output for the journal as a figure at =
> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when =
> the images were indexed color, the up sampling was done by taking each =
> pixel and subdividing it into smaller pixels.  Thus the pixelation of =
> the original was maintained and the editor did not like that.  

Then the editor does not understand the subject he is dealing with here.
You cant upscale and interpolate an image just so it looks nice when its blown up.
This is generation of information that you did not originally record.

If it were a table of numbers (which of course it actually is!)
he would never ask you to fill in the gaps between rows and columns with new numbers!!!
NEVER!


> What they =
> wanted done was to convert the images to RGB and then upsample.  In that =
> case (or grayscale), Photoshop does an interpolation making a smoother =
> looking zoomed in view, but that is a change in the data.

Yes, that thais plain WRONG.

>  The index =
> version is actually more accurate although less pleasing.

But it is correct.

> I plan to =
> argue that this is not necessary or desirable, but I did not know that =
> Photoshop (but not ImageJ) makes this distinction and thought others =
> might want to know it happens.  Does anyone know why index vs. RGB =
> should matter to the up sampling algorithm.  THanks- Dave

The matter is essentially moot, as what should happen these days is this:

You supply a Tiny little thumbnail image that is the same size in pixels at the correct DPI
as the journal will finally print in the obsolete paper copy
and as a garbled and corrupted jpeg compressed image in the PDF version online.
This image is a place holder, and is just used as a pointer to the real image data:

There is no way to show a 2000x2000 confocal image on a sheet of paper, so don't bother trying.
But you can make an annotated thumbnail image that point you to the real thing:

You will supply in the text or supplemental info the hyperlink to the location of the real original data file captured by the
microscope software. Then the reader clicks that link, down loads the real image data (from your webserver, or some central database), and
analyses it themselves (using your openly published analysis method - maybe an imageJ macro or plugin)

JBC already has this built in to their website:
they have the online data viewer, where your real images go into a database (powered by OME)
http://jcb-dataviewer.rupress.org/
so you can browse the data online, and also download it.

We need an image database .... like the genomic data bases or other biological public open databases
where we can put our images and screens so that others can get the data when we publish it,
or even before. This is a requirement of publication, and in out discipline we usually ignore it.
This is very very very _WRONG_!!!!

If your editor demands that you provide RGB JPEG images of a certain DPI and number of pixels
then hes/she is trapped in the early 20th Century,
and you should educate them that things have moved on.

What they should be asking you for is the original data files
so they can put them in their own version of the JCB Data Viewer
and so they can give them to the referees of the paper!

Any opinions?

Dan




Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Well, I'm sorry but I think you should reverse your polarity!

If your research is numerical you don't publish vast tables of numbers,
you present the data as graphs, histograms and statistical summaries
with standard deviations, etc.  That way it can be understood.  If it's
available to you, you place the numerical data in an archive, and I'm
all in favour of that.  Exactly the same thing applies to digital
images.  You could try to publish your images as tables of ASCII numbers
rather than pictorially (elderly folk like me will remember that early
image analysis programs such as Semper used to save images in that
format) but I defy you to get a journal to accept them!  

                                                 Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Daniel James White
Sent: Friday, 9 September 2011 6:35 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Dave and Listers,

I grow more polarized on this topic....

On Sep 9, 2011, at 7:02 AM, CONFOCALMICROSCOPY automatic digest system
wrote:

>
> Date:    Thu, 8 Sep 2011 11:24:52 +0100
> From:    David Knecht <[hidden email]>
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
> *****
>
> I have been forced back into this issue in helping someone get an

image =
> taken on our microscopes through a journal editor.  The images were =
> fairly low resolution.

THe data is the data... there no no sensible reason to try to make it
"look nicer"

>  They were manipulated in Photoshop, imported =
> into Canvas, text added and then output for the journal as a figure at
=
> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop,
when =
> the images were indexed color, the up sampling was done by taking each
=
> pixel and subdividing it into smaller pixels.  Thus the pixelation of
=
> the original was maintained and the editor did not like that.  

Then the editor does not understand the subject he is dealing with here.

You cant upscale and interpolate an image just so it looks nice when its
blown up.
This is generation of information that you did not originally record.

If it were a table of numbers (which of course it actually is!)
he would never ask you to fill in the gaps between rows and columns with
new numbers!!!
NEVER!


> What they =
> wanted done was to convert the images to RGB and then upsample.  In
that =
> case (or grayscale), Photoshop does an interpolation making a smoother
=
> looking zoomed in view, but that is a change in the data.

Yes, that thais plain WRONG.

>  The index =
> version is actually more accurate although less pleasing.

But it is correct.

> I plan to =
> argue that this is not necessary or desirable, but I did not know that
=
> Photoshop (but not ImageJ) makes this distinction and thought others =
> might want to know it happens.  Does anyone know why index vs. RGB =
> should matter to the up sampling algorithm.  THanks- Dave

The matter is essentially moot, as what should happen these days is
this:

You supply a Tiny little thumbnail image that is the same size in pixels
at the correct DPI
as the journal will finally print in the obsolete paper copy
and as a garbled and corrupted jpeg compressed image in the PDF version
online.
This image is a place holder, and is just used as a pointer to the real
image data:

There is no way to show a 2000x2000 confocal image on a sheet of paper,
so don't bother trying.
But you can make an annotated thumbnail image that point you to the real
thing:

You will supply in the text or supplemental info the hyperlink to the
location of the real original data file captured by the
microscope software. Then the reader clicks that link, down loads the
real image data (from your webserver, or some central database), and
analyses it themselves (using your openly published analysis method -
maybe an imageJ macro or plugin)

JBC already has this built in to their website:
they have the online data viewer, where your real images go into a
database (powered by OME)
http://jcb-dataviewer.rupress.org/
so you can browse the data online, and also download it.

We need an image database .... like the genomic data bases or other
biological public open databases
where we can put our images and screens so that others can get the data
when we publish it,
or even before. This is a requirement of publication, and in out
discipline we usually ignore it.
This is very very very _WRONG_!!!!

If your editor demands that you provide RGB JPEG images of a certain DPI
and number of pixels
then hes/she is trapped in the early 20th Century,
and you should educate them that things have moved on.

What they should be asking you for is the original data files
so they can put them in their own version of the JCB Data Viewer
and so they can give them to the referees of the paper!

Any opinions?

Dan




Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ
(Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging
Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Mark Cannell]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy

I don't think that's quite right, surely it's the final production magnification that determines whether the original structures are resolved at 300 DPI?

FWIW, just last week had a Wiley production compartment (in China!) complain that one of my pics was not 300 DPI (they said it was 258 DPI !). The funny thing is that the images were all 300 DPI when I sent them off (would not have passed their brainless image quality check tool otherwise).  They didn't like the new copy I sent either (still 300 DPI) and asked me to send them the original data so they could process it correctly for me!   The issue was almost certainly that they did not like the fact that you could actually see individual pixels in the image. I on the other hand thought it quite acceptable -but what do I know about it... Since the processing of the image took about many many lines of IDL code I politely declined their kind offer to reprocess.

Cheers Mark
 
On 9/09/2011, at 9:28 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Brian,
>
>         In my view you are indeed missing something.  Nyquist sampling is ~2.3 pixels per resel.  At 300dpi these pixels would be 85µm in size, so you couldn't see structures that are resolved in the image.  A 512x512 image would be 43 mm or 1.7" square.  
>
>   The aim of publication is communication, so to communicate your results you need to present them in a way that they can be understood when published in a journal.  In any case, a confocal image is just a series of sample points - Nyquist theory assumes that these will be presented as sine waves, not blocks.  So mapping your samples into a higher-resolution space is in fact the correct thing to do.  By all means put your original data into an archive, and I wish more journals would provide this facility (though equally I can understand why they don't).  
>
>  If you are my age (or even half my age) you would once have recorded your images on 35mm film.  Did you insist on publishing them at that size (24x36mm)?  Of course not, you printed the negatives at the required size.  That's changing the data a LOT more than bicubic resampling.  
>
>                                       Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
> Sent: Friday, 9 September 2011 3:32 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is the request to upload an image that has increased Dpi?
> For this I would open the image in Photoshop, open image size dialogue box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
> It seems to me that resampling a scientific image using an algorithm such as bicubic is interpolating pixels and therefore creating new data where it did not exist before.
> Am I missing something?
>
> Brian Armstrong PhD
> Assistant Research Professor
> Light Microscopy Core
> Beckman Research Institute
> City of Hope
> 1500 East Duarte Road
> Duarte, CA 91010
> 626-256-4673 x62872
>
> Light Microscopy Core Facility
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: Thursday, September 08, 2011 5:15 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> David,
>
>              I suppose it's partly Photoshop trying to guess what users
> want, and partly a matter of what's computationally possible.  If you
> are talking about 8-bit indexed color (ie grayscale with a palette
> added) there is no way you can you can apply any of the sophisticated
> resampling algorithms to it.  Turning it into RGB and resampling is
> indeed wrong, very wrong, what you must do is turn it into grayscale and
> resample, then re-apply the color palette.  The best resampling
> algorithm is bicubic interpolation (see my chapter in the Pawley book,
> where I compare algorithms).  Note that when converting the image to
> grayscale you must do it by applying a grayscale palette while
> maintaining indices, not using  'closest match' which will give you
> nonsense.
>
>              Generally I don't use Photoshop, I use Paint Shop Pro, but
> the basic steps should be similar.  Generate a gray-scale image with all
> 256 values present, and save the palette as a palette file (I call it
> lin_grey).  Open the user's image and save the palette as another
> palette file.  Now replace the palette, maintaining indices, by
> lin_grey.  You now have a grayscale image which you can scale
> effectively.  Scale with bicubic interpolation to the required value,
> then re-apply the original palette (again, of course, maintaining
> indices).
>
>                Is this kosher? Absolutely, 100%.  Your image IS a
> gray-scale image, the palette is just an add-on.  It's no different from
> enlarging a negative in a darkroom enlarger.  In the end your picture
> will be represented in the printed page by an array of dots, between 120
> and 300 to the inch, depending on the quality of the journal.  Your goal
> is to get the data to convert accurately to this representation.
>
>                                         Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of David Knecht
> Sent: Thursday, 8 September 2011 8:25 PM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have been forced back into this issue in helping someone get an image
> taken on our microscopes through a journal editor.  The images were
> fairly low resolution.  They were manipulated in Photoshop, imported
> into Canvas, text added and then output for the journal as a figure at
> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
> the images were indexed color, the up sampling was done by taking each
> pixel and subdividing it into smaller pixels.  Thus the pixelation of
> the original was maintained and the editor did not like that.  What they
> wanted done was to convert the images to RGB and then upsample.  In that
> case (or grayscale), Photoshop does an interpolation making a smoother
> looking zoomed in view, but that is a change in the data.  The index
> version is actually more accurate although less pleasing. I plan to
> argue that this is not necessary or desirable, but I did not know that
> Photoshop (but not ImageJ) makes this distinction and thought others
> might want to know it happens.  Does anyone know why index vs. RGB
> should matter to the up sampling algorithm.  THanks- Dave
>
>
> On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:
>
>> Well, as George McNamara has suggested many times on the list the
> image you should send in the "mean image", (an example of the images
> collected).
>> I would predict that this is rarely the case and that most images sent
> to journals are extreme examples of the best possible image collected
> and corrected.
>> However, sending the entire image set (in Gigabytes or even Terabytes)
> to the journal seems impractical. Perhaps instead one could acquire the
> entire data set by e-mailing the author.
>>
>>
>> Brian D Armstrong PhD
>> Light Microscopy Core Manager
>> Beckman Research Institute
>> City of Hope
>> Dept of Neuroscience
>> 1450 E Duarte Rd
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
> ing/Pages/default.aspx
>> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Andreas
> Bruckbauer
>> Sent: Tuesday, July 13, 2010 2:14 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> Dear all,
>> so far this discussion mainly focusses on "the  image" but one image
> alone can not represent the data. Think about the data set  represented
> by a distribution  which might be gaussian, so you would  need  at
> least the mean and  the  width of the  distribution. What i want to say
> is that you will need to do a quantification and then show  an image
> representing  the mean and show a histogram which gives the reader a
> clue about how the data  is  distributed. There might be more than  one
> variable which is  important for the  analysis and of  course you should
> do an idependend repeat of the  experiment.
>> So what would you suggest, sending all the original image files to the
> data bank?
>> I still think that rather than re-analysing other scientists data a
> repeat of the  experiment in another lab is more important, there are a
> lot of thinks apart  from data analysis and representation which can go
> wrong. However i see the point that when the paper is about image
> analysis to  provide the original files to let other groups  repreat the
> analysis.
>>
>> best wishes
>>
>> Andreas
>>
>>
>>
>>
>> -----Original Message-----
>> From: Daniel James White <[hidden email]>
>> To: [hidden email]
>> Sent: Tue, 13 Jul 2010 9:01
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> Dear Jerry,
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> cheers for adding your valuable input to this discussion.
>>
>>
>>
>>
>> Its been a long and interesting one so far.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest
> system wrote:
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Date:    Mon, 12 Jul 2010 12:47:51 -0500
>>
>>
>>
>>
>>> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>>
>>
>>
>>
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>>
>>
>>
>>>
>>
>>
>>
>>
>>> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>>
>>
>>
>>
>>> <html>
>>
>>
>>
>>
>>> <head>
>>
>>
>>
>>
>>> <meta content="text/html; charset=windows-1252"
>>
>>
>>
>>
>>> http-equiv="Content-Type">
>>
>>
>>
>>
>>> </head>
>>
>>
>>
>>
>>> <body bgcolor="#ffffff" text="#000000">
>>
>>
>>
>>
>>> Hi All,<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> maybe you might like to turn off HTML text formatting in your email
> client when
>>
>>
>>
>>
>> you post to the lists
>>
>>
>>
>>
>> in this case its not so distracting but sometimes emails to this an
> other list
>>
>>
>>
>>
>> as unreadable in digest mode
>>
>>
>>
>>
>> due to the thousands of html tags.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> I'm a little late in addressing this issue, but the PPI/DPI part of
>>
>>
>>
>>
>>> this conversation is misleading.  What I mean to say is that the
> real
>>
>>
>>
>>
>>> issue may not be resolution, but reproduction.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> A very good point.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Images that are sent to
>>
>>
>>
>>
>>> publishers with the full dynamic range of 0 - 255 pixel values may
>>
>>
>>
>>
>>> likely be reproduced with no details at the bright end (240 - 255),
> and
>>
>>
>>
>>
>>> the dark end (0 - 20).  Printing presses cannot reproduce detail
> within
>>
>>
>>
>>
>>> these tonal ranges because of limitations with dropping ink on paper
>>
>>
>>
>>
>>> without A) having the drop not stick when the tonal values are
> bright
>>
>>
>>
>>
>>> and B) having the inks blend into each other through capillary
> action
>>
>>
>>
>>
>>> at the darkest values.  This phenomenon gets worse when the paper is
>>
>>
>>
>>
>>> lower quality (e.g., "Science").<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> So what we are saying is maybe summarised as follows:
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Small thumbnail images in print can only every be just that - pointers
> to go and
>>
>>
>>
>>
>> look at the real digital image on some online source.
>>
>>
>>
>>
>> No one can hope to reach quantitative conclusions from a small print
> image.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> We can probably consign print images on paper to the heap of old
> technologies,
>>
>>
>>
>>
>> which are no longer relevant to the work we do.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Images in PDF files "could" be useful, but not if they are lossy
> compressed and
>>
>>
>>
>>
>> missing meta data, which they usually are.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Color reproduction is generally worse because primary and secondary
>>
>>
>>
>>
>>> colors are used to show experimental evidence.  These colors are not
>>
>>
>>
>>
>>> always within the range of printing presses, and so these tend to
> print
>>
>>
>>
>>
>>> blobs sans detail when color choices are not appropriate for
> publishing
>>
>>
>>
>>
>>> onto paper.<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> To make matters worse....
>>
>>
>>
>>
>> fors most peoples eyes, blue is much fainter than red and green, with
> green
>>
>>
>>
>>
>> often being precieved as brightest.
>>
>>
>>
>>
>> So forget even semi  quantitative comparisons of intensity there.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> In any case we dont print in red green and blue, so these are silly
> choices for
>>
>>
>>
>>
>> a print version of figures.
>>
>>
>>
>>
>> The print colour space would be better, Magenta, yellow and cyan....
> but still
>>
>>
>>
>>
>> for our quantitative purposed not good.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Screen (LCD/CRT) colours are more similar to out eyes trichromatic
> response.
>>
>>
>>
>>
>> That why they are red green and blue... but the blue looks darker then
> green
>>
>>
>>
>>
>> problem still persists.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> More often than not, the issue of whether or not details can be
>>
>>
>>
>>
>>> resolved by eye on a printed page is not that of how many pixels
> exist
>>
>>
>>
>>
>>> in the submitted image, but in how effectively tones and colors were
>>
>>
>>
>>
>>> fitted to the printing press.  This is especially true if the pdf
> image
>>
>>
>>
>>
>>> at non-zoomed, computer screen resolution reveals desired details,
> but
>>
>>
>>
>>
>>> the printed page does not:  it is more likely that a 133 line per
> inch
>>
>>
>>
>>
>>> screened image appearing in publication has more resolution than the
>>
>>
>>
>>
>>> computer screen (often figured at an average of 90 pixels per inch,
>>
>>
>>
>>
>>> with 72 pixels being the "old" standard).  The color and tonal range
> of
>>
>>
>>
>>
>>> reproduction of a computer screen is greater than on a printing
> press.<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Indeed it is, and so is a better visualisation tool...
>>
>>
>>
>>
>> but to get the most out of it, we also need to be smart.
>>
>>
>>
>>
>> We cant compare the brightness of blue and green due to our
> physiology,
>>
>>
>>
>>
>> even if the screen is properly calibrated (usually not the case).
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> So, we should not ry to do that on screen or in print.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Our eyes are much better at discriminating greyscale brightness scale,
>
>>
>>
>>
>>
>> but actually we are sill pretty bad at that.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> see this example of where we are easily fooled when trying to compare
> grey scale
>>
>>
>>
>>
>> brightness.
>>
>>
>>
>>
>> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> The situation with colouyr is more complicated, and also beset with
> pitfalle
>>
>>
>>
>>
>> due to the way our optical system works... it tries to find contrast
> in any
>>
>>
>>
>>
>> scene,
>>
>>
>>
>>
>> and even generates contrast or colour differences that are not there
> (but gave
>>
>>
>>
>>
>> us an evolutionary advantage in picking fruit)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> See the spiral image at the bottom f this colocalisation tutorial,
>>
>>
>>
>>
>> which shows us that we should be careful when interpreting colour
> merge multi
>>
>>
>>
>>
>> channel images.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> you can draw the spiral pattern yourself in imageJ with the marco
> script there,
>>
>>
>>
>>
>> and prove to yourself that your eyes lie to you.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Greyscale images can be made interpretable in a semi quantitative
> manner by
>>
>>
>>
>>
>> using for instance the fire colour look up table.
>>
>>
>>
>>
>> Each bright colour represents some intensity value, so its easier to
> compare
>>
>>
>>
>>
>> intensities over and between images.
>>
>>
>>
>>
>> Physicists and chemists do this all the time, and think we are mad for
> showing
>>
>>
>>
>>
>> DAPI staining  in black to Blue - because "thats what it looks like"
>>
>>
>>
>>
>> They are right , we are mad to do that.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Like others before this email, I believe that it is best that
>>
>>
>>
>>
>>> scientists take the task of reproduction as much in their own hands
> as
>>
>>
>>
>>
>>> possible so that the outcome can be controlled.  The image is a
>>
>>
>>
>>
>>> reproduction of what was once under a microscope, and it behooves
> the
>>
>>
>>
>>
>>> researcher to make that appear as close to the original
> representation
>>
>>
>>
>>
>>> as possible.  <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> I'm not sure that this is ever going to be a helpful way to approach
> the
>>
>>
>>
>>
>> problem.
>>
>>
>>
>>
>> No matter how carefully you set out your images and send them to the
> publisher,
>>
>>
>>
>>
>> you have no control and what they do with them next. None.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> The only way around this is to have the images in the paper act only
> as
>>
>>
>>
>>
>> thumbnails
>>
>>
>>
>>
>> which point to the original image data file(s) on an online
> repository,
>>
>>
>>
>>
>> like the JCB image viewer or our wished for public biological image
> database...
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> An image is not the sample, it always contain much less info that the
> sample
>>
>>
>>
>>
>> did.
>>
>>
>>
>>
>> The trick is to keep the useful info as it passes through the scope
> lenses,
>>
>>
>>
>>
>> the detector, and the computer you your brain/eyes.
>>
>>
>>
>>
>> I agree, an image is a representation of the info that made it through
> the
>>
>>
>>
>>
>> microscope to you.
>>
>>
>>
>>
>> Trying to represent it "faithfully" and as  it "truly looks" are both
> missing
>>
>>
>>
>>
>> the mark.
>>
>>
>>
>>
>> The image is usually degraded by the blur of the Point spread function
> / OTF
>>
>>
>>
>>
>> and by various sources of noise.
>>
>>
>>
>>
>> Thus, the image is an artifact in of itself. What we want to know
> about is the
>>
>>
>>
>>
>> sample.
>>
>>
>>
>>
>> The images contains info from the sample in a degraded  and incomplete
> from.
>>
>>
>>
>>
>> We have to work around that, and not pretend that the image fully
> represents the
>>
>>
>>
>>
>> sample.
>>
>>
>>
>>
>> It does not.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> As far as images being data points, this also is true, and these are
> to
>>
>>
>>
>>
>>> remain unaltered (unless flatfield correcting, background
> subtracting,
>>
>>
>>
>>
>>> etc) for measuring.  A faithful representation of that image when
>>
>>
>>
>>
>>> reproduced is another matter altogether.<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Indeed it is, and i think there is even no such thing as a faithful
>>
>>
>>
>>
>> representation of the Sample as an image,
>>
>>
>>
>>
>> so the faithful representation of the image is also then something to
> think
>>
>>
>>
>>
>> about.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> I think what matters is How you represent the image data....
>>
>>
>>
>>
>> in some cases an illustration like Hooke's drawing of cells might even
> be better
>>
>>
>>
>>
>> than a digital representation,
>>
>>
>>
>>
>> if you want to get a certain message across to the reader. Original
> digital
>>
>>
>>
>>
>> image data available too of course online.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> cheers
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dan
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Cheers,<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Jerry Sedgewick<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>>
>>
>>
>> Senior Microscopist / Image Visualisation, Processing and Analysis
>>
>>
>>
>>
>> Light Microscopy and Image Processing Facilities
>>
>>
>>
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>>
>>
>>
>>
>> Pfotenhauerstrasse 108
>>
>>
>>
>>
>> 01307 DRESDEN
>>
>>
>>
>>
>> Germany
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> [phone] (German Mobile)
>>
>>
>>
>>
>> [phone] (Work phone at MPI-CBG)
>>
>>
>>
>>
>> [phone] (Fax MPI-CBG LMF)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> http://www.bioimagexd.net   BioImageXD
>>
>>
>>
>>
>> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries
> Included)
>>
>>
>>
>>
>> http://www.chalkie.org.uk       Dan's Homepages
>>
>>
>>
>>
>> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>>
>>
>>
>>
>> dan (at) chalkie.org.uk
>>
>>
>>
>>
>> ( white (at) mpi-cbg.de )
>>
>>
>>
>>
>>
>> ---------------------------------------------------------------------
>> SECURITY/CONFIDENTIALITY WARNING:
>> This message and any attachments are intended solely for the
> individual or entity to which they are addressed. This communication may
> contain information that is privileged, confidential, or exempt from
> disclosure under applicable law (e.g., personal health information,
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>> ---------------------------------------------------------------------
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Mark,

    Journals generally want the images to be 300dpi at their final reproduction size.  I agree that doesn't entirely make sense but even as the editor of a microscopy journal I can't persuade production to see things any other way.  So if you just change the declared resolution in the header to 300 dpi on a 512x512 image it will come out very small and you'll see nothing.  As to your case, if you have checked the TIFF header and it says 300dpi I can't quite see how anyone could claim it's 258 dpi!  However, I cannot see any merit in seeing individual pixels except in very special cases.  As I said in a previous post - and as you know very well - the Nyquist criterion is based on the highest sampling rate being reproduced as a sine wave.  If it appears as blocks (square waves) there are axiomatically higher frequencies present.  

        We have to consider the human visual system, too.  This has a built in and very powerful edge-recognition algorithm.  This in turn means that if pixels are visible to actual data often is not.

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Friday, 9 September 2011 7:08 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Guy

I don't think that's quite right, surely it's the final production magnification that determines whether the original structures are resolved at 300 DPI?

FWIW, just last week had a Wiley production compartment (in China!) complain that one of my pics was not 300 DPI (they said it was 258 DPI !). The funny thing is that the images were all 300 DPI when I sent them off (would not have passed their brainless image quality check tool otherwise).  They didn't like the new copy I sent either (still 300 DPI) and asked me to send them the original data so they could process it correctly for me!   The issue was almost certainly that they did not like the fact that you could actually see individual pixels in the image. I on the other hand thought it quite acceptable -but what do I know about it... Since the processing of the image took about many many lines of IDL code I politely declined their kind offer to reprocess.

Cheers Mark
 
On 9/09/2011, at 9:28 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Brian,
>
>         In my view you are indeed missing something.  Nyquist sampling is ~2.3 pixels per resel.  At 300dpi these pixels would be 85µm in size, so you couldn't see structures that are resolved in the image.  A 512x512 image would be 43 mm or 1.7" square.  
>
>   The aim of publication is communication, so to communicate your results you need to present them in a way that they can be understood when published in a journal.  In any case, a confocal image is just a series of sample points - Nyquist theory assumes that these will be presented as sine waves, not blocks.  So mapping your samples into a higher-resolution space is in fact the correct thing to do.  By all means put your original data into an archive, and I wish more journals would provide this facility (though equally I can understand why they don't).  
>
>  If you are my age (or even half my age) you would once have recorded your images on 35mm film.  Did you insist on publishing them at that size (24x36mm)?  Of course not, you printed the negatives at the required size.  That's changing the data a LOT more than bicubic resampling.  
>
>                                       Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Armstrong, Brian
> Sent: Friday, 9 September 2011 3:32 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is the request to upload an image that has increased Dpi?
> For this I would open the image in Photoshop, open image size dialogue box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
> It seems to me that resampling a scientific image using an algorithm such as bicubic is interpolating pixels and therefore creating new data where it did not exist before.
> Am I missing something?
>
> Brian Armstrong PhD
> Assistant Research Professor
> Light Microscopy Core
> Beckman Research Institute
> City of Hope
> 1500 East Duarte Road
> Duarte, CA 91010
> 626-256-4673 x62872
>
> Light Microscopy Core Facility
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: Thursday, September 08, 2011 5:15 AM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> David,
>
>              I suppose it's partly Photoshop trying to guess what users
> want, and partly a matter of what's computationally possible.  If you
> are talking about 8-bit indexed color (ie grayscale with a palette
> added) there is no way you can you can apply any of the sophisticated
> resampling algorithms to it.  Turning it into RGB and resampling is
> indeed wrong, very wrong, what you must do is turn it into grayscale and
> resample, then re-apply the color palette.  The best resampling
> algorithm is bicubic interpolation (see my chapter in the Pawley book,
> where I compare algorithms).  Note that when converting the image to
> grayscale you must do it by applying a grayscale palette while
> maintaining indices, not using  'closest match' which will give you
> nonsense.
>
>              Generally I don't use Photoshop, I use Paint Shop Pro, but
> the basic steps should be similar.  Generate a gray-scale image with all
> 256 values present, and save the palette as a palette file (I call it
> lin_grey).  Open the user's image and save the palette as another
> palette file.  Now replace the palette, maintaining indices, by
> lin_grey.  You now have a grayscale image which you can scale
> effectively.  Scale with bicubic interpolation to the required value,
> then re-apply the original palette (again, of course, maintaining
> indices).
>
>                Is this kosher? Absolutely, 100%.  Your image IS a
> gray-scale image, the palette is just an add-on.  It's no different from
> enlarging a negative in a darkroom enlarger.  In the end your picture
> will be represented in the printed page by an array of dots, between 120
> and 300 to the inch, depending on the quality of the journal.  Your goal
> is to get the data to convert accurately to this representation.
>
>                                         Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>             Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of David Knecht
> Sent: Thursday, 8 September 2011 8:25 PM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have been forced back into this issue in helping someone get an image
> taken on our microscopes through a journal editor.  The images were
> fairly low resolution.  They were manipulated in Photoshop, imported
> into Canvas, text added and then output for the journal as a figure at
> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
> the images were indexed color, the up sampling was done by taking each
> pixel and subdividing it into smaller pixels.  Thus the pixelation of
> the original was maintained and the editor did not like that.  What they
> wanted done was to convert the images to RGB and then upsample.  In that
> case (or grayscale), Photoshop does an interpolation making a smoother
> looking zoomed in view, but that is a change in the data.  The index
> version is actually more accurate although less pleasing. I plan to
> argue that this is not necessary or desirable, but I did not know that
> Photoshop (but not ImageJ) makes this distinction and thought others
> might want to know it happens.  Does anyone know why index vs. RGB
> should matter to the up sampling algorithm.  THanks- Dave
>
>
> On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:
>
>> Well, as George McNamara has suggested many times on the list the
> image you should send in the "mean image", (an example of the images
> collected).
>> I would predict that this is rarely the case and that most images sent
> to journals are extreme examples of the best possible image collected
> and corrected.
>> However, sending the entire image set (in Gigabytes or even Terabytes)
> to the journal seems impractical. Perhaps instead one could acquire the
> entire data set by e-mailing the author.
>>
>>
>> Brian D Armstrong PhD
>> Light Microscopy Core Manager
>> Beckman Research Institute
>> City of Hope
>> Dept of Neuroscience
>> 1450 E Duarte Rd
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
> ing/Pages/default.aspx
>> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Andreas
> Bruckbauer
>> Sent: Tuesday, July 13, 2010 2:14 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> Dear all,
>> so far this discussion mainly focusses on "the  image" but one image
> alone can not represent the data. Think about the data set  represented
> by a distribution  which might be gaussian, so you would  need  at
> least the mean and  the  width of the  distribution. What i want to say
> is that you will need to do a quantification and then show  an image
> representing  the mean and show a histogram which gives the reader a
> clue about how the data  is  distributed. There might be more than  one
> variable which is  important for the  analysis and of  course you should
> do an idependend repeat of the  experiment.
>> So what would you suggest, sending all the original image files to the
> data bank?
>> I still think that rather than re-analysing other scientists data a
> repeat of the  experiment in another lab is more important, there are a
> lot of thinks apart  from data analysis and representation which can go
> wrong. However i see the point that when the paper is about image
> analysis to  provide the original files to let other groups  repreat the
> analysis.
>>
>> best wishes
>>
>> Andreas
>>
>>
>>
>>
>> -----Original Message-----
>> From: Daniel James White <[hidden email]>
>> To: [hidden email]
>> Sent: Tue, 13 Jul 2010 9:01
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> Dear Jerry,
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> cheers for adding your valuable input to this discussion.
>>
>>
>>
>>
>> Its been a long and interesting one so far.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest
> system wrote:
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Date:    Mon, 12 Jul 2010 12:47:51 -0500
>>
>>
>>
>>
>>> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>>
>>
>>
>>
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>>
>>
>>
>>>
>>
>>
>>
>>
>>> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>>
>>
>>
>>
>>> <html>
>>
>>
>>
>>
>>> <head>
>>
>>
>>
>>
>>> <meta content="text/html; charset=windows-1252"
>>
>>
>>
>>
>>> http-equiv="Content-Type">
>>
>>
>>
>>
>>> </head>
>>
>>
>>
>>
>>> <body bgcolor="#ffffff" text="#000000">
>>
>>
>>
>>
>>> Hi All,<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> maybe you might like to turn off HTML text formatting in your email
> client when
>>
>>
>>
>>
>> you post to the lists
>>
>>
>>
>>
>> in this case its not so distracting but sometimes emails to this an
> other list
>>
>>
>>
>>
>> as unreadable in digest mode
>>
>>
>>
>>
>> due to the thousands of html tags.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> I'm a little late in addressing this issue, but the PPI/DPI part of
>>
>>
>>
>>
>>> this conversation is misleading.  What I mean to say is that the
> real
>>
>>
>>
>>
>>> issue may not be resolution, but reproduction.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> A very good point.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Images that are sent to
>>
>>
>>
>>
>>> publishers with the full dynamic range of 0 - 255 pixel values may
>>
>>
>>
>>
>>> likely be reproduced with no details at the bright end (240 - 255),
> and
>>
>>
>>
>>
>>> the dark end (0 - 20).  Printing presses cannot reproduce detail
> within
>>
>>
>>
>>
>>> these tonal ranges because of limitations with dropping ink on paper
>>
>>
>>
>>
>>> without A) having the drop not stick when the tonal values are
> bright
>>
>>
>>
>>
>>> and B) having the inks blend into each other through capillary
> action
>>
>>
>>
>>
>>> at the darkest values.  This phenomenon gets worse when the paper is
>>
>>
>>
>>
>>> lower quality (e.g., "Science").<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> So what we are saying is maybe summarised as follows:
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Small thumbnail images in print can only every be just that - pointers
> to go and
>>
>>
>>
>>
>> look at the real digital image on some online source.
>>
>>
>>
>>
>> No one can hope to reach quantitative conclusions from a small print
> image.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> We can probably consign print images on paper to the heap of old
> technologies,
>>
>>
>>
>>
>> which are no longer relevant to the work we do.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Images in PDF files "could" be useful, but not if they are lossy
> compressed and
>>
>>
>>
>>
>> missing meta data, which they usually are.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Color reproduction is generally worse because primary and secondary
>>
>>
>>
>>
>>> colors are used to show experimental evidence.  These colors are not
>>
>>
>>
>>
>>> always within the range of printing presses, and so these tend to
> print
>>
>>
>>
>>
>>> blobs sans detail when color choices are not appropriate for
> publishing
>>
>>
>>
>>
>>> onto paper.<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> To make matters worse....
>>
>>
>>
>>
>> fors most peoples eyes, blue is much fainter than red and green, with
> green
>>
>>
>>
>>
>> often being precieved as brightest.
>>
>>
>>
>>
>> So forget even semi  quantitative comparisons of intensity there.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> In any case we dont print in red green and blue, so these are silly
> choices for
>>
>>
>>
>>
>> a print version of figures.
>>
>>
>>
>>
>> The print colour space would be better, Magenta, yellow and cyan....
> but still
>>
>>
>>
>>
>> for our quantitative purposed not good.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Screen (LCD/CRT) colours are more similar to out eyes trichromatic
> response.
>>
>>
>>
>>
>> That why they are red green and blue... but the blue looks darker then
> green
>>
>>
>>
>>
>> problem still persists.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> More often than not, the issue of whether or not details can be
>>
>>
>>
>>
>>> resolved by eye on a printed page is not that of how many pixels
> exist
>>
>>
>>
>>
>>> in the submitted image, but in how effectively tones and colors were
>>
>>
>>
>>
>>> fitted to the printing press.  This is especially true if the pdf
> image
>>
>>
>>
>>
>>> at non-zoomed, computer screen resolution reveals desired details,
> but
>>
>>
>>
>>
>>> the printed page does not:  it is more likely that a 133 line per
> inch
>>
>>
>>
>>
>>> screened image appearing in publication has more resolution than the
>>
>>
>>
>>
>>> computer screen (often figured at an average of 90 pixels per inch,
>>
>>
>>
>>
>>> with 72 pixels being the "old" standard).  The color and tonal range
> of
>>
>>
>>
>>
>>> reproduction of a computer screen is greater than on a printing
> press.<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Indeed it is, and so is a better visualisation tool...
>>
>>
>>
>>
>> but to get the most out of it, we also need to be smart.
>>
>>
>>
>>
>> We cant compare the brightness of blue and green due to our
> physiology,
>>
>>
>>
>>
>> even if the screen is properly calibrated (usually not the case).
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> So, we should not ry to do that on screen or in print.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Our eyes are much better at discriminating greyscale brightness scale,
>
>>
>>
>>
>>
>> but actually we are sill pretty bad at that.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> see this example of where we are easily fooled when trying to compare
> grey scale
>>
>>
>>
>>
>> brightness.
>>
>>
>>
>>
>> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> The situation with colouyr is more complicated, and also beset with
> pitfalle
>>
>>
>>
>>
>> due to the way our optical system works... it tries to find contrast
> in any
>>
>>
>>
>>
>> scene,
>>
>>
>>
>>
>> and even generates contrast or colour differences that are not there
> (but gave
>>
>>
>>
>>
>> us an evolutionary advantage in picking fruit)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> See the spiral image at the bottom f this colocalisation tutorial,
>>
>>
>>
>>
>> which shows us that we should be careful when interpreting colour
> merge multi
>>
>>
>>
>>
>> channel images.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> you can draw the spiral pattern yourself in imageJ with the marco
> script there,
>>
>>
>>
>>
>> and prove to yourself that your eyes lie to you.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Greyscale images can be made interpretable in a semi quantitative
> manner by
>>
>>
>>
>>
>> using for instance the fire colour look up table.
>>
>>
>>
>>
>> Each bright colour represents some intensity value, so its easier to
> compare
>>
>>
>>
>>
>> intensities over and between images.
>>
>>
>>
>>
>> Physicists and chemists do this all the time, and think we are mad for
> showing
>>
>>
>>
>>
>> DAPI staining  in black to Blue - because "thats what it looks like"
>>
>>
>>
>>
>> They are right , we are mad to do that.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> Like others before this email, I believe that it is best that
>>
>>
>>
>>
>>> scientists take the task of reproduction as much in their own hands
> as
>>
>>
>>
>>
>>> possible so that the outcome can be controlled.  The image is a
>>
>>
>>
>>
>>> reproduction of what was once under a microscope, and it behooves
> the
>>
>>
>>
>>
>>> researcher to make that appear as close to the original
> representation
>>
>>
>>
>>
>>> as possible.  <br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> I'm not sure that this is ever going to be a helpful way to approach
> the
>>
>>
>>
>>
>> problem.
>>
>>
>>
>>
>> No matter how carefully you set out your images and send them to the
> publisher,
>>
>>
>>
>>
>> you have no control and what they do with them next. None.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> The only way around this is to have the images in the paper act only
> as
>>
>>
>>
>>
>> thumbnails
>>
>>
>>
>>
>> which point to the original image data file(s) on an online
> repository,
>>
>>
>>
>>
>> like the JCB image viewer or our wished for public biological image
> database...
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> An image is not the sample, it always contain much less info that the
> sample
>>
>>
>>
>>
>> did.
>>
>>
>>
>>
>> The trick is to keep the useful info as it passes through the scope
> lenses,
>>
>>
>>
>>
>> the detector, and the computer you your brain/eyes.
>>
>>
>>
>>
>> I agree, an image is a representation of the info that made it through
> the
>>
>>
>>
>>
>> microscope to you.
>>
>>
>>
>>
>> Trying to represent it "faithfully" and as  it "truly looks" are both
> missing
>>
>>
>>
>>
>> the mark.
>>
>>
>>
>>
>> The image is usually degraded by the blur of the Point spread function
> / OTF
>>
>>
>>
>>
>> and by various sources of noise.
>>
>>
>>
>>
>> Thus, the image is an artifact in of itself. What we want to know
> about is the
>>
>>
>>
>>
>> sample.
>>
>>
>>
>>
>> The images contains info from the sample in a degraded  and incomplete
> from.
>>
>>
>>
>>
>> We have to work around that, and not pretend that the image fully
> represents the
>>
>>
>>
>>
>> sample.
>>
>>
>>
>>
>> It does not.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> As far as images being data points, this also is true, and these are
> to
>>
>>
>>
>>
>>> remain unaltered (unless flatfield correcting, background
> subtracting,
>>
>>
>>
>>
>>> etc) for measuring.  A faithful representation of that image when
>>
>>
>>
>>
>>> reproduced is another matter altogether.<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Indeed it is, and i think there is even no such thing as a faithful
>>
>>
>>
>>
>> representation of the Sample as an image,
>>
>>
>>
>>
>> so the faithful representation of the image is also then something to
> think
>>
>>
>>
>>
>> about.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> I think what matters is How you represent the image data....
>>
>>
>>
>>
>> in some cases an illustration like Hooke's drawing of cells might even
> be better
>>
>>
>>
>>
>> than a digital representation,
>>
>>
>>
>>
>> if you want to get a certain message across to the reader. Original
> digital
>>
>>
>>
>>
>> image data available too of course online.
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> cheers
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dan
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Cheers,<br>
>>
>>
>>
>>
>>> <br>
>>
>>
>>
>>
>>> Jerry Sedgewick<br>
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> Dr. Daniel James White BSc. (Hons.) PhD
>>
>>
>>
>>
>> Senior Microscopist / Image Visualisation, Processing and Analysis
>>
>>
>>
>>
>> Light Microscopy and Image Processing Facilities
>>
>>
>>
>>
>> Max Planck Institute of Molecular Cell Biology and Genetics
>>
>>
>>
>>
>> Pfotenhauerstrasse 108
>>
>>
>>
>>
>> 01307 DRESDEN
>>
>>
>>
>>
>> Germany
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> [phone] (German Mobile)
>>
>>
>>
>>
>> [phone] (Work phone at MPI-CBG)
>>
>>
>>
>>
>> [phone] (Fax MPI-CBG LMF)
>>
>>
>>
>>
>>
>>
>>
>>
>>
>> http://www.bioimagexd.net   BioImageXD
>>
>>
>>
>>
>> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries
> Included)
>>
>>
>>
>>
>> http://www.chalkie.org.uk       Dan's Homepages
>>
>>
>>
>>
>> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>>
>>
>>
>>
>> dan (at) chalkie.org.uk
>>
>>
>>
>>
>> ( white (at) mpi-cbg.de )
>>
>>
>>
>>
>>
>> ---------------------------------------------------------------------
>> SECURITY/CONFIDENTIALITY WARNING:
>> This message and any attachments are intended solely for the
> individual or entity to which they are addressed. This communication may
> contain information that is privileged, confidential, or exempt from
> disclosure under applicable law (e.g., personal health information,
> research data, financial information). Because this e-mail has been sent
> without encryption, individuals other than the intended recipient may be
> able to view the information, forward it to others or tamper with the
> information without the knowledge or consent of the sender. If you are
> not the intended recipient, or the employee or person responsible for
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> do not wish to receive further communications via e-mail, please reply
> to this message and inform the sender that you do not wish to receive
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>> ---------------------------------------------------------------------
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)

[Dmitry Sokolov-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,

I am afraid that the confusion about Nyquist sampling is still there.
Nyquist-Shannon theorem is about full digital description of analogue
signal with highest possible frequency. This is conventionally applied in
its pure form to critical dimension (CD) imaging where the techniques are
facing their physical limitations.

The same theorem applied to non-CD imaging and perception of digital
images refers to number of pixels per feature of interest rather than
resel. Features of interest in optical imaging of a human face and
electron imaging of a letter on a coin for example will have negligibly
low spatial frequency compared to theoretical. The features will be
successfully resolved when presented by more than 2 pixels in the image. I
usually suggest 2-10 pixels per a feature as the lower limit to my
students. Pixels in this case are either physical pixels on the screen or
a resels on paper (0.1mm). The actual number of pixels per feature depends
on the size of the overall structure to be presented in the image. A
feature as small as 1/1000 of the width of the image will require zooming
to be perceived or resolved by the human eye.

The same magic number 1000 is involved in printing and computer interface
matters. 3" 300 dpi image will contain about a 1000 pixels across.
Conventional displays and data projectors have slightly more than a 1000
pixels along the screen. You may be probably agree now that 1000 pixels
images are the current standard for scanning and digital imaging. Bigger
number of pixels will create slightly better looking images according to
Nyquist criteria on the expense of higher acquisition time and lower
intensity of signal according to "Triangle of Frustration" principles:
http://confocal-manawatu.pbworks.com/w/page/37083554/Triangle%20of%20Frustration

I believe that 300 dpi is a big misleading requirement to the images. 3"
images in the text and 7" image on a cover of a journal will have
different number of pixels at the same pixel density. PPI at an image
printed size advised as a reference is what I would vote for.

Concerning the resampling of pixelated images, it's no nice at all. In my
old student time no data would be allowed without explanation and
validation of all of the processing steps and procedures. Pixel to resel
or pixel to pixel ratio should be 1x1 in ideal case for any kind of
presentation. Publishing on a cover would require high-resolution primary
data to remain scientific. Those are the ideal requirements. The actual
situation with presentation and publishing of visual scientific data is
however drastically different.

Here is the permalink at MIAWiki on the topic to those who is interested
in developing the "final" Wiki page:
http://confocal-manawatu.pbworks.com/w/page/45307622/Nyquist%20Sampling%20for%20non-CD%20Imaging

Thank you,
Dmitry

On Fri, September 9, 2011 9:27 pm, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Mark,
>
>     Journals generally want the images to be 300dpi at their final
> reproduction size.  I agree that doesn't entirely make sense but even
> as the editor of a microscopy journal I can't persuade production to
> see things any other way.  So if you just change the declared
> resolution in the header to 300 dpi on a 512x512 image it will come
> out very small and you'll see nothing.  As to your case, if you have
> checked the TIFF header and it says 300dpi I can't quite see how
> anyone could claim it's 258 dpi!  However, I cannot see any merit in
> seeing individual pixels except in very special cases.  As I said in a
> previous post - and as you know very well - the Nyquist criterion is
> based on the highest sampling rate being reproduced as a sine wave.
> If it appears as blocks (square waves) there are axiomatically higher
> frequencies present.
>
> We have to consider the human visual system, too.  This has a built in
> and very powerful edge-recognition algorithm.  This in turn means that if
> pixels are visible to actual data often is not.
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: Friday, 9 September 2011 7:08 PM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy
>
> I don't think that's quite right, surely it's the final production
> magnification that determines whether the original structures are resolved
> at 300 DPI?
>
> FWIW, just last week had a Wiley production compartment (in China!)
> complain that one of my pics was not 300 DPI (they said it was 258 DPI !).
> The funny thing is that the images were all 300 DPI when I sent them off
> (would not have passed their brainless image quality check tool
> otherwise).  They didn't like the new copy I sent either (still 300 DPI)
> and asked me to send them the original data so they could process it
> correctly for me!   The issue was almost certainly that they did not like
> the fact that you could actually see individual pixels in the image. I on
> the other hand thought it quite acceptable -but what do I know about it...
> Since the processing of the image took about many many lines of IDL code I
> politely declined their kind offer to reprocess.
>
> Cheers Mark
>
> On 9/09/2011, at 9:28 AM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Brian,
>>
>>         In my view you are indeed missing something.  Nyquist sampling
>> is ~2.3 pixels per resel.  At 300dpi these pixels would be 85µm
>> in size, so you couldn't see structures that are resolved in the
>> image.  A 512x512 image would be 43 mm or 1.7" square.
>>
>>   The aim of publication is communication, so to communicate your
>> results you need to present them in a way that they can be
>> understood when published in a journal.  In any case, a confocal
>> image is just a series of sample points - Nyquist theory assumes
>> that these will be presented as sine waves, not blocks.  So mapping
>> your samples into a higher-resolution space is in fact the correct
>> thing to do.  By all means put your original data into an archive,
>> and I wish more journals would provide this facility (though equally
>> I can understand why they don't).
>>
>>  If you are my age (or even half my age) you would once have recorded
>> your images on 35mm film.  Did you insist on publishing them at that
>> size (24x36mm)?  Of course not, you printed the negatives at the
>> required size.  That's changing the data a LOT more than bicubic
>> resampling.
>>
>>                                       Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Armstrong, Brian
>> Sent: Friday, 9 September 2011 3:32 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Is the request to upload an image that has increased Dpi?
>> For this I would open the image in Photoshop, open image size dialogue
>> box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
>> It seems to me that resampling a scientific image using an algorithm
>> such as bicubic is interpolating pixels and therefore creating new data
>> where it did not exist before.
>> Am I missing something?
>>
>> Brian Armstrong PhD
>> Assistant Research Professor
>> Light Microscopy Core
>> Beckman Research Institute
>> City of Hope
>> 1500 East Duarte Road
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
>> Light Microscopy Core Facility
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Guy Cox
>> Sent: Thursday, September 08, 2011 5:15 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> David,
>>
>>              I suppose it's partly Photoshop trying to guess what users
>> want, and partly a matter of what's computationally possible.  If you
>> are talking about 8-bit indexed color (ie grayscale with a palette
>> added) there is no way you can you can apply any of the sophisticated
>> resampling algorithms to it.  Turning it into RGB and resampling is
>> indeed wrong, very wrong, what you must do is turn it into grayscale and
>> resample, then re-apply the color palette.  The best resampling
>> algorithm is bicubic interpolation (see my chapter in the Pawley book,
>> where I compare algorithms).  Note that when converting the image to
>> grayscale you must do it by applying a grayscale palette while
>> maintaining indices, not using  'closest match' which will give you
>> nonsense.
>>
>>              Generally I don't use Photoshop, I use Paint Shop Pro, but
>> the basic steps should be similar.  Generate a gray-scale image with all
>> 256 values present, and save the palette as a palette file (I call it
>> lin_grey).  Open the user's image and save the palette as another
>> palette file.  Now replace the palette, maintaining indices, by
>> lin_grey.  You now have a grayscale image which you can scale
>> effectively.  Scale with bicubic interpolation to the required value,
>> then re-apply the original palette (again, of course, maintaining
>> indices).
>>
>>                Is this kosher? Absolutely, 100%.  Your image IS a
>> gray-scale image, the palette is just an add-on.  It's no different from
>> enlarging a negative in a darkroom enlarger.  In the end your picture
>> will be represented in the printed page by an array of dots, between 120
>> and 300 to the inch, depending on the quality of the journal.  Your goal
>> is to get the data to convert accurately to this representation.
>>
>>                                         Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of David Knecht
>> Sent: Thursday, 8 September 2011 8:25 PM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I have been forced back into this issue in helping someone get an image
>> taken on our microscopes through a journal editor.  The images were
>> fairly low resolution.  They were manipulated in Photoshop, imported
>> into Canvas, text added and then output for the journal as a figure at
>> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
>> the images were indexed color, the up sampling was done by taking each
>> pixel and subdividing it into smaller pixels.  Thus the pixelation of
>> the original was maintained and the editor did not like that.  What they
>> wanted done was to convert the images to RGB and then upsample.  In that
>> case (or grayscale), Photoshop does an interpolation making a smoother
>> looking zoomed in view, but that is a change in the data.  The index
>> version is actually more accurate although less pleasing. I plan to
>> argue that this is not necessary or desirable, but I did not know that
>> Photoshop (but not ImageJ) makes this distinction and thought others
>> might want to know it happens.  Does anyone know why index vs. RGB
>> should matter to the up sampling algorithm.  THanks- Dave
>>
>>
>> On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:
>>
>>> Well, as George McNamara has suggested many times on the list the
>> image you should send in the "mean image", (an example of the images
>> collected).
>>> I would predict that this is rarely the case and that most images sent
>> to journals are extreme examples of the best possible image collected
>> and corrected.
>>> However, sending the entire image set (in Gigabytes or even Terabytes)
>> to the journal seems impractical. Perhaps instead one could acquire the
>> entire data set by e-mailing the author.
>>>
>>>
>>> Brian D Armstrong PhD
>>> Light Microscopy Core Manager
>>> Beckman Research Institute
>>> City of Hope
>>> Dept of Neuroscience
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
>> ing/Pages/default.aspx
>>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Andreas
>> Bruckbauer
>>> Sent: Tuesday, July 13, 2010 2:14 AM
>>> To: [hidden email]
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>> Dear all,
>>> so far this discussion mainly focusses on "the  image" but one image
>> alone can not represent the data. Think about the data set  represented
>> by a distribution  which might be gaussian, so you would  need  at
>> least the mean and  the  width of the  distribution. What i want to say
>> is that you will need to do a quantification and then show  an image
>> representing  the mean and show a histogram which gives the reader a
>> clue about how the data  is  distributed. There might be more than  one
>> variable which is  important for the  analysis and of  course you should
>> do an idependend repeat of the  experiment.
>>> So what would you suggest, sending all the original image files to the
>> data bank?
>>> I still think that rather than re-analysing other scientists data a
>> repeat of the  experiment in another lab is more important, there are a
>> lot of thinks apart  from data analysis and representation which can go
>> wrong. However i see the point that when the paper is about image
>> analysis to  provide the original files to let other groups  repreat the
>> analysis.
>>>
>>> best wishes
>>>
>>> Andreas
>>>
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Daniel James White <[hidden email]>
>>> To: [hidden email]
>>> Sent: Tue, 13 Jul 2010 9:01
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>> Dear Jerry,
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> cheers for adding your valuable input to this discussion.
>>>
>>>
>>>
>>>
>>> Its been a long and interesting one so far.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest
>> system wrote:
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Date:    Mon, 12 Jul 2010 12:47:51 -0500
>>>
>>>
>>>
>>>
>>>> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>>>
>>>
>>>
>>>
>>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>>
>>>
>>>
>>>>
>>>
>>>
>>>
>>>
>>>> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>>>
>>>
>>>
>>>
>>>> <html>
>>>
>>>
>>>
>>>
>>>> <head>
>>>
>>>
>>>
>>>
>>>> <meta content="text/html; charset=windows-1252"
>>>
>>>
>>>
>>>
>>>> http-equiv="Content-Type">
>>>
>>>
>>>
>>>
>>>> </head>
>>>
>>>
>>>
>>>
>>>> <body bgcolor="#ffffff" text="#000000">
>>>
>>>
>>>
>>>
>>>> Hi All,<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> maybe you might like to turn off HTML text formatting in your email
>> client when
>>>
>>>
>>>
>>>
>>> you post to the lists
>>>
>>>
>>>
>>>
>>> in this case its not so distracting but sometimes emails to this an
>> other list
>>>
>>>
>>>
>>>
>>> as unreadable in digest mode
>>>
>>>
>>>
>>>
>>> due to the thousands of html tags.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> I'm a little late in addressing this issue, but the PPI/DPI part of
>>>
>>>
>>>
>>>
>>>> this conversation is misleading.  What I mean to say is that the
>> real
>>>
>>>
>>>
>>>
>>>> issue may not be resolution, but reproduction.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> A very good point.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Images that are sent to
>>>
>>>
>>>
>>>
>>>> publishers with the full dynamic range of 0 - 255 pixel values may
>>>
>>>
>>>
>>>
>>>> likely be reproduced with no details at the bright end (240 - 255),
>> and
>>>
>>>
>>>
>>>
>>>> the dark end (0 - 20).  Printing presses cannot reproduce detail
>> within
>>>
>>>
>>>
>>>
>>>> these tonal ranges because of limitations with dropping ink on paper
>>>
>>>
>>>
>>>
>>>> without A) having the drop not stick when the tonal values are
>> bright
>>>
>>>
>>>
>>>
>>>> and B) having the inks blend into each other through capillary
>> action
>>>
>>>
>>>
>>>
>>>> at the darkest values.  This phenomenon gets worse when the paper is
>>>
>>>
>>>
>>>
>>>> lower quality (e.g., "Science").<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> So what we are saying is maybe summarised as follows:
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Small thumbnail images in print can only every be just that - pointers
>> to go and
>>>
>>>
>>>
>>>
>>> look at the real digital image on some online source.
>>>
>>>
>>>
>>>
>>> No one can hope to reach quantitative conclusions from a small print
>> image.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> We can probably consign print images on paper to the heap of old
>> technologies,
>>>
>>>
>>>
>>>
>>> which are no longer relevant to the work we do.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Images in PDF files "could" be useful, but not if they are lossy
>> compressed and
>>>
>>>
>>>
>>>
>>> missing meta data, which they usually are.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Color reproduction is generally worse because primary and secondary
>>>
>>>
>>>
>>>
>>>> colors are used to show experimental evidence.  These colors are not
>>>
>>>
>>>
>>>
>>>> always within the range of printing presses, and so these tend to
>> print
>>>
>>>
>>>
>>>
>>>> blobs sans detail when color choices are not appropriate for
>> publishing
>>>
>>>
>>>
>>>
>>>> onto paper.<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> To make matters worse....
>>>
>>>
>>>
>>>
>>> fors most peoples eyes, blue is much fainter than red and green, with
>> green
>>>
>>>
>>>
>>>
>>> often being precieved as brightest.
>>>
>>>
>>>
>>>
>>> So forget even semi  quantitative comparisons of intensity there.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> In any case we dont print in red green and blue, so these are silly
>> choices for
>>>
>>>
>>>
>>>
>>> a print version of figures.
>>>
>>>
>>>
>>>
>>> The print colour space would be better, Magenta, yellow and cyan....
>> but still
>>>
>>>
>>>
>>>
>>> for our quantitative purposed not good.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Screen (LCD/CRT) colours are more similar to out eyes trichromatic
>> response.
>>>
>>>
>>>
>>>
>>> That why they are red green and blue... but the blue looks darker then
>> green
>>>
>>>
>>>
>>>
>>> problem still persists.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> More often than not, the issue of whether or not details can be
>>>
>>>
>>>
>>>
>>>> resolved by eye on a printed page is not that of how many pixels
>> exist
>>>
>>>
>>>
>>>
>>>> in the submitted image, but in how effectively tones and colors were
>>>
>>>
>>>
>>>
>>>> fitted to the printing press.  This is especially true if the pdf
>> image
>>>
>>>
>>>
>>>
>>>> at non-zoomed, computer screen resolution reveals desired details,
>> but
>>>
>>>
>>>
>>>
>>>> the printed page does not:  it is more likely that a 133 line per
>> inch
>>>
>>>
>>>
>>>
>>>> screened image appearing in publication has more resolution than the
>>>
>>>
>>>
>>>
>>>> computer screen (often figured at an average of 90 pixels per inch,
>>>
>>>
>>>
>>>
>>>> with 72 pixels being the "old" standard).  The color and tonal range
>> of
>>>
>>>
>>>
>>>
>>>> reproduction of a computer screen is greater than on a printing
>> press.<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Indeed it is, and so is a better visualisation tool...
>>>
>>>
>>>
>>>
>>> but to get the most out of it, we also need to be smart.
>>>
>>>
>>>
>>>
>>> We cant compare the brightness of blue and green due to our
>> physiology,
>>>
>>>
>>>
>>>
>>> even if the screen is properly calibrated (usually not the case).
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> So, we should not ry to do that on screen or in print.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Our eyes are much better at discriminating greyscale brightness scale,
>>
>>>
>>>
>>>
>>>
>>> but actually we are sill pretty bad at that.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> see this example of where we are easily fooled when trying to compare
>> grey scale
>>>
>>>
>>>
>>>
>>> brightness.
>>>
>>>
>>>
>>>
>>> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> The situation with colouyr is more complicated, and also beset with
>> pitfalle
>>>
>>>
>>>
>>>
>>> due to the way our optical system works... it tries to find contrast
>> in any
>>>
>>>
>>>
>>>
>>> scene,
>>>
>>>
>>>
>>>
>>> and even generates contrast or colour differences that are not there
>> (but gave
>>>
>>>
>>>
>>>
>>> us an evolutionary advantage in picking fruit)
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> See the spiral image at the bottom f this colocalisation tutorial,
>>>
>>>
>>>
>>>
>>> which shows us that we should be careful when interpreting colour
>> merge multi
>>>
>>>
>>>
>>>
>>> channel images.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> you can draw the spiral pattern yourself in imageJ with the marco
>> script there,
>>>
>>>
>>>
>>>
>>> and prove to yourself that your eyes lie to you.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Greyscale images can be made interpretable in a semi quantitative
>> manner by
>>>
>>>
>>>
>>>
>>> using for instance the fire colour look up table.
>>>
>>>
>>>
>>>
>>> Each bright colour represents some intensity value, so its easier to
>> compare
>>>
>>>
>>>
>>>
>>> intensities over and between images.
>>>
>>>
>>>
>>>
>>> Physicists and chemists do this all the time, and think we are mad for
>> showing
>>>
>>>
>>>
>>>
>>> DAPI staining  in black to Blue - because "thats what it looks like"
>>>
>>>
>>>
>>>
>>> They are right , we are mad to do that.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Like others before this email, I believe that it is best that
>>>
>>>
>>>
>>>
>>>> scientists take the task of reproduction as much in their own hands
>> as
>>>
>>>
>>>
>>>
>>>> possible so that the outcome can be controlled.  The image is a
>>>
>>>
>>>
>>>
>>>> reproduction of what was once under a microscope, and it behooves
>> the
>>>
>>>
>>>
>>>
>>>> researcher to make that appear as close to the original
>> representation
>>>
>>>
>>>
>>>
>>>> as possible.  <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> I'm not sure that this is ever going to be a helpful way to approach
>> the
>>>
>>>
>>>
>>>
>>> problem.
>>>
>>>
>>>
>>>
>>> No matter how carefully you set out your images and send them to the
>> publisher,
>>>
>>>
>>>
>>>
>>> you have no control and what they do with them next. None.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> The only way around this is to have the images in the paper act only
>> as
>>>
>>>
>>>
>>>
>>> thumbnails
>>>
>>>
>>>
>>>
>>> which point to the original image data file(s) on an online
>> repository,
>>>
>>>
>>>
>>>
>>> like the JCB image viewer or our wished for public biological image
>> database...
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> An image is not the sample, it always contain much less info that the
>> sample
>>>
>>>
>>>
>>>
>>> did.
>>>
>>>
>>>
>>>
>>> The trick is to keep the useful info as it passes through the scope
>> lenses,
>>>
>>>
>>>
>>>
>>> the detector, and the computer you your brain/eyes.
>>>
>>>
>>>
>>>
>>> I agree, an image is a representation of the info that made it through
>> the
>>>
>>>
>>>
>>>
>>> microscope to you.
>>>
>>>
>>>
>>>
>>> Trying to represent it "faithfully" and as  it "truly looks" are both
>> missing
>>>
>>>
>>>
>>>
>>> the mark.
>>>
>>>
>>>
>>>
>>> The image is usually degraded by the blur of the Point spread function
>> / OTF
>>>
>>>
>>>
>>>
>>> and by various sources of noise.
>>>
>>>
>>>
>>>
>>> Thus, the image is an artifact in of itself. What we want to know
>> about is the
>>>
>>>
>>>
>>>
>>> sample.
>>>
>>>
>>>
>>>
>>> The images contains info from the sample in a degraded  and incomplete
>> from.
>>>
>>>
>>>
>>>
>>> We have to work around that, and not pretend that the image fully
>> represents the
>>>
>>>
>>>
>>>
>>> sample.
>>>
>>>
>>>
>>>
>>> It does not.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> As far as images being data points, this also is true, and these are
>> to
>>>
>>>
>>>
>>>
>>>> remain unaltered (unless flatfield correcting, background
>> subtracting,
>>>
>>>
>>>
>>>
>>>> etc) for measuring.  A faithful representation of that image when
>>>
>>>
>>>
>>>
>>>> reproduced is another matter altogether.<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Indeed it is, and i think there is even no such thing as a faithful
>>>
>>>
>>>
>>>
>>> representation of the Sample as an image,
>>>
>>>
>>>
>>>
>>> so the faithful representation of the image is also then something to
>> think
>>>
>>>
>>>
>>>
>>> about.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> I think what matters is How you represent the image data....
>>>
>>>
>>>
>>>
>>> in some cases an illustration like Hooke's drawing of cells might even
>> be better
>>>
>>>
>>>
>>>
>>> than a digital representation,
>>>
>>>
>>>
>>>
>>> if you want to get a certain message across to the reader. Original
>> digital
>>>
>>>
>>>
>>>
>>> image data available too of course online.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> cheers
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Dan
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Cheers,<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Jerry Sedgewick<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Dr. Daniel James White BSc. (Hons.) PhD
>>>
>>>
>>>
>>>
>>> Senior Microscopist / Image Visualisation, Processing and Analysis
>>>
>>>
>>>
>>>
>>> Light Microscopy and Image Processing Facilities
>>>
>>>
>>>
>>>
>>> Max Planck Institute of Molecular Cell Biology and Genetics
>>>
>>>
>>>
>>>
>>> Pfotenhauerstrasse 108
>>>
>>>
>>>
>>>
>>> 01307 DRESDEN
>>>
>>>
>>>
>>>
>>> Germany
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> [phone] (German Mobile)
>>>
>>>
>>>
>>>
>>> [phone] (Work phone at MPI-CBG)
>>>
>>>
>>>
>>>
>>> [phone] (Fax MPI-CBG LMF)
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> http://www.bioimagexd.net   BioImageXD
>>>
>>>
>>>
>>>
>>> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries
>> Included)
>>>
>>>
>>>
>>>
>>> http://www.chalkie.org.uk       Dan's Homepages
>>>
>>>
>>>
>>>
>>> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>>>
>>>
>>>
>>>
>>> dan (at) chalkie.org.uk
>>>
>>>
>>>
>>>
>>> ( white (at) mpi-cbg.de )
>>>
>>>
>>>
>>>
>>>
>>> ---------------------------------------------------------------------
>>> SECURITY/CONFIDENTIALITY WARNING:
>>> This message and any attachments are intended solely for the
>> individual or entity to which they are addressed. This communication may
>> contain information that is privileged, confidential, or exempt from
>> disclosure under applicable law (e.g., personal health information,
>> research data, financial information). Because this e-mail has been sent
>> without encryption, individuals other than the intended recipient may be
>> able to view the information, forward it to others or tamper with the
>> information without the knowledge or consent of the sender. If you are
>> not the intended recipient, or the employee or person responsible for
>> delivering the message to the intended recipient, any dissemination,
>> distribution or copying of the communication is strictly prohibited. If
>> you received the communication in error, please notify the sender
>> immediately by replying to this message and deleting the message and any
>> accompanying files from your system. If, due to the security risks, you
>> do not wish to receive further communications via e-mail, please reply
>> to this message and inform the sender that you do not wish to receive
>> further e-mail from the sender.
>>> ---------------------------------------------------------------------
>>
>> Dr. David Knecht
>> Department of Molecular and Cell Biology
>> Co-head Flow Cytometry and Confocal Microscopy Facility
>> U-3125
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)
>

--
Dr. Dmitry Sokolov
Institute of Fundamental Sciences
Massey University, Palmerston North
New Zealand

[Guy Cox-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dmitry,

  This list is not about imaging faces, it is about confocal microscopy.  We have one criterion only, the Rayleigh resolution.  I advise students to, where possible, acquire images with 3 pixels within the Rayleigh resolution of their objective.  This is slight oversampling, but it is justified for two reasons: (1) it makes details at the resolution limit easier to see, and (2) it allows for mild (VERY mild) filtering to remove noise without affecting resolution.  Any further oversampling just bleaches the specimen without giving any further information.  So now we have acquired our data.  We are scientists, not photographers, so now we have two things to do, ANALYSE our data (which can involve a whole string of operations) and PRESENT our data.  In the presentation phase we cannot expect a journal to magically give its readers the value of each pixel on the printed page - the image is printed with a halftone screen, for a start, and also it will have a particular gamma so that the original data is not reproduced linearly (actually your computer screen doesn't have a linear gamma either, though high-end imaging programs will allow to adjust this).  

                If we print a 35mm negative for publication, we'll enlarge it to 3" wide for a 1-column image or 7" wide for a full-page presentation.  We'll also pick a grade of paper which gives the correct contrast for the purpose.  Working with digital images we should do exactly the same.  This is not changing the data (which I hope is safely stored somewhere) it is presenting it.  Likewise, when I analyse intra-membrane particles I don't present every measurement, I publish a histogram, with mean values and standard deviations.  But of course I still have the measurement of every particle.  The reader does not expect to see the raw data - in the case of a digital image on paper that is impossible, and in the case of a particle analysis it would be incomprehensible - he or she expects to be presented with what the data reveals.

                                                  Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dmitry Sokolov
Sent: Friday, 9 September 2011 10:21 PM
To: [hidden email]
Subject: Re: Preparing figures for publication --PPI vs DPI

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear All,

I am afraid that the confusion about Nyquist sampling is still there.
Nyquist-Shannon theorem is about full digital description of analogue
signal with highest possible frequency. This is conventionally applied in
its pure form to critical dimension (CD) imaging where the techniques are
facing their physical limitations.

The same theorem applied to non-CD imaging and perception of digital
images refers to number of pixels per feature of interest rather than
resel. Features of interest in optical imaging of a human face and
electron imaging of a letter on a coin for example will have negligibly
low spatial frequency compared to theoretical. The features will be
successfully resolved when presented by more than 2 pixels in the image. I
usually suggest 2-10 pixels per a feature as the lower limit to my
students. Pixels in this case are either physical pixels on the screen or
a resels on paper (0.1mm). The actual number of pixels per feature depends
on the size of the overall structure to be presented in the image. A
feature as small as 1/1000 of the width of the image will require zooming
to be perceived or resolved by the human eye.

The same magic number 1000 is involved in printing and computer interface
matters. 3" 300 dpi image will contain about a 1000 pixels across.
Conventional displays and data projectors have slightly more than a 1000
pixels along the screen. You may be probably agree now that 1000 pixels
images are the current standard for scanning and digital imaging. Bigger
number of pixels will create slightly better looking images according to
Nyquist criteria on the expense of higher acquisition time and lower
intensity of signal according to "Triangle of Frustration" principles:
http://confocal-manawatu.pbworks.com/w/page/37083554/Triangle%20of%20Frustration

I believe that 300 dpi is a big misleading requirement to the images. 3"
images in the text and 7" image on a cover of a journal will have
different number of pixels at the same pixel density. PPI at an image
printed size advised as a reference is what I would vote for.

Concerning the resampling of pixelated images, it's no nice at all. In my
old student time no data would be allowed without explanation and
validation of all of the processing steps and procedures. Pixel to resel
or pixel to pixel ratio should be 1x1 in ideal case for any kind of
presentation. Publishing on a cover would require high-resolution primary
data to remain scientific. Those are the ideal requirements. The actual
situation with presentation and publishing of visual scientific data is
however drastically different.

Here is the permalink at MIAWiki on the topic to those who is interested
in developing the "final" Wiki page:
http://confocal-manawatu.pbworks.com/w/page/45307622/Nyquist%20Sampling%20for%20non-CD%20Imaging

Thank you,
Dmitry

On Fri, September 9, 2011 9:27 pm, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Mark,
>
>     Journals generally want the images to be 300dpi at their final
> reproduction size.  I agree that doesn't entirely make sense but even
> as the editor of a microscopy journal I can't persuade production to
> see things any other way.  So if you just change the declared
> resolution in the header to 300 dpi on a 512x512 image it will come
> out very small and you'll see nothing.  As to your case, if you have
> checked the TIFF header and it says 300dpi I can't quite see how
> anyone could claim it's 258 dpi!  However, I cannot see any merit in
> seeing individual pixels except in very special cases.  As I said in a
> previous post - and as you know very well - the Nyquist criterion is
> based on the highest sampling rate being reproduced as a sine wave.
> If it appears as blocks (square waves) there are axiomatically higher
> frequencies present.
>
> We have to consider the human visual system, too.  This has a built in
> and very powerful edge-recognition algorithm.  This in turn means that if
> pixels are visible to actual data often is not.
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: Friday, 9 September 2011 7:08 PM
> To: [hidden email]
> Subject: Re: Preparing figures for publication --PPI vs DPI
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy
>
> I don't think that's quite right, surely it's the final production
> magnification that determines whether the original structures are resolved
> at 300 DPI?
>
> FWIW, just last week had a Wiley production compartment (in China!)
> complain that one of my pics was not 300 DPI (they said it was 258 DPI !).
> The funny thing is that the images were all 300 DPI when I sent them off
> (would not have passed their brainless image quality check tool
> otherwise).  They didn't like the new copy I sent either (still 300 DPI)
> and asked me to send them the original data so they could process it
> correctly for me!   The issue was almost certainly that they did not like
> the fact that you could actually see individual pixels in the image. I on
> the other hand thought it quite acceptable -but what do I know about it...
> Since the processing of the image took about many many lines of IDL code I
> politely declined their kind offer to reprocess.
>
> Cheers Mark
>
> On 9/09/2011, at 9:28 AM, Guy Cox wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Brian,
>>
>>         In my view you are indeed missing something.  Nyquist sampling
>> is ~2.3 pixels per resel.  At 300dpi these pixels would be 85µm
>> in size, so you couldn't see structures that are resolved in the
>> image.  A 512x512 image would be 43 mm or 1.7" square.
>>
>>   The aim of publication is communication, so to communicate your
>> results you need to present them in a way that they can be
>> understood when published in a journal.  In any case, a confocal
>> image is just a series of sample points - Nyquist theory assumes
>> that these will be presented as sine waves, not blocks.  So mapping
>> your samples into a higher-resolution space is in fact the correct
>> thing to do.  By all means put your original data into an archive,
>> and I wish more journals would provide this facility (though equally
>> I can understand why they don't).
>>
>>  If you are my age (or even half my age) you would once have recorded
>> your images on 35mm film.  Did you insist on publishing them at that
>> size (24x36mm)?  Of course not, you printed the negatives at the
>> required size.  That's changing the data a LOT more than bicubic
>> resampling.
>>
>>                                       Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Armstrong, Brian
>> Sent: Friday, 9 September 2011 3:32 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Is the request to upload an image that has increased Dpi?
>> For this I would open the image in Photoshop, open image size dialogue
>> box, type in resolution 300dpi, UNCHECK Resample image, and choose OK.
>> It seems to me that resampling a scientific image using an algorithm
>> such as bicubic is interpolating pixels and therefore creating new data
>> where it did not exist before.
>> Am I missing something?
>>
>> Brian Armstrong PhD
>> Assistant Research Professor
>> Light Microscopy Core
>> Beckman Research Institute
>> City of Hope
>> 1500 East Duarte Road
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
>> Light Microscopy Core Facility
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Guy Cox
>> Sent: Thursday, September 08, 2011 5:15 AM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> David,
>>
>>              I suppose it's partly Photoshop trying to guess what users
>> want, and partly a matter of what's computationally possible.  If you
>> are talking about 8-bit indexed color (ie grayscale with a palette
>> added) there is no way you can you can apply any of the sophisticated
>> resampling algorithms to it.  Turning it into RGB and resampling is
>> indeed wrong, very wrong, what you must do is turn it into grayscale and
>> resample, then re-apply the color palette.  The best resampling
>> algorithm is bicubic interpolation (see my chapter in the Pawley book,
>> where I compare algorithms).  Note that when converting the image to
>> grayscale you must do it by applying a grayscale palette while
>> maintaining indices, not using  'closest match' which will give you
>> nonsense.
>>
>>              Generally I don't use Photoshop, I use Paint Shop Pro, but
>> the basic steps should be similar.  Generate a gray-scale image with all
>> 256 values present, and save the palette as a palette file (I call it
>> lin_grey).  Open the user's image and save the palette as another
>> palette file.  Now replace the palette, maintaining indices, by
>> lin_grey.  You now have a grayscale image which you can scale
>> effectively.  Scale with bicubic interpolation to the required value,
>> then re-apply the original palette (again, of course, maintaining
>> indices).
>>
>>                Is this kosher? Absolutely, 100%.  Your image IS a
>> gray-scale image, the palette is just an add-on.  It's no different from
>> enlarging a negative in a darkroom enlarger.  In the end your picture
>> will be represented in the printed page by an array of dots, between 120
>> and 300 to the inch, depending on the quality of the journal.  Your goal
>> is to get the data to convert accurately to this representation.
>>
>>                                         Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>     http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>             Mobile [phone]
>> ______________________________________________
>>      http://www.guycox.net
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of David Knecht
>> Sent: Thursday, 8 September 2011 8:25 PM
>> To: [hidden email]
>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I have been forced back into this issue in helping someone get an image
>> taken on our microscopes through a journal editor.  The images were
>> fairly low resolution.  They were manipulated in Photoshop, imported
>> into Canvas, text added and then output for the journal as a figure at
>> 300dpi TIFF's in photoshop.  What we noticed is that in Photoshop, when
>> the images were indexed color, the up sampling was done by taking each
>> pixel and subdividing it into smaller pixels.  Thus the pixelation of
>> the original was maintained and the editor did not like that.  What they
>> wanted done was to convert the images to RGB and then upsample.  In that
>> case (or grayscale), Photoshop does an interpolation making a smoother
>> looking zoomed in view, but that is a change in the data.  The index
>> version is actually more accurate although less pleasing. I plan to
>> argue that this is not necessary or desirable, but I did not know that
>> Photoshop (but not ImageJ) makes this distinction and thought others
>> might want to know it happens.  Does anyone know why index vs. RGB
>> should matter to the up sampling algorithm.  THanks- Dave
>>
>>
>> On Jul 13, 2010, at 4:59 PM, Armstrong, Brian wrote:
>>
>>> Well, as George McNamara has suggested many times on the list the
>> image you should send in the "mean image", (an example of the images
>> collected).
>>> I would predict that this is rarely the case and that most images sent
>> to journals are extreme examples of the best possible image collected
>> and corrected.
>>> However, sending the entire image set (in Gigabytes or even Terabytes)
>> to the journal seems impractical. Perhaps instead one could acquire the
>> entire data set by e-mailing the author.
>>>
>>>
>>> Brian D Armstrong PhD
>>> Light Microscopy Core Manager
>>> Beckman Research Institute
>>> City of Hope
>>> Dept of Neuroscience
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>> http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
>> ing/Pages/default.aspx
>>> From: Confocal Microscopy List
>> [mailto:[hidden email]] On Behalf Of Andreas
>> Bruckbauer
>>> Sent: Tuesday, July 13, 2010 2:14 AM
>>> To: [hidden email]
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>> Dear all,
>>> so far this discussion mainly focusses on "the  image" but one image
>> alone can not represent the data. Think about the data set  represented
>> by a distribution  which might be gaussian, so you would  need  at
>> least the mean and  the  width of the  distribution. What i want to say
>> is that you will need to do a quantification and then show  an image
>> representing  the mean and show a histogram which gives the reader a
>> clue about how the data  is  distributed. There might be more than  one
>> variable which is  important for the  analysis and of  course you should
>> do an idependend repeat of the  experiment.
>>> So what would you suggest, sending all the original image files to the
>> data bank?
>>> I still think that rather than re-analysing other scientists data a
>> repeat of the  experiment in another lab is more important, there are a
>> lot of thinks apart  from data analysis and representation which can go
>> wrong. However i see the point that when the paper is about image
>> analysis to  provide the original files to let other groups  repreat the
>> analysis.
>>>
>>> best wishes
>>>
>>> Andreas
>>>
>>>
>>>
>>>
>>> -----Original Message-----
>>> From: Daniel James White <[hidden email]>
>>> To: [hidden email]
>>> Sent: Tue, 13 Jul 2010 9:01
>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>> Dear Jerry,
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> cheers for adding your valuable input to this discussion.
>>>
>>>
>>>
>>>
>>> Its been a long and interesting one so far.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> On Jul 13, 2010, at 7:01 AM, CONFOCALMICROSCOPY automatic digest
>> system wrote:
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Date:    Mon, 12 Jul 2010 12:47:51 -0500
>>>
>>>
>>>
>>>
>>>> From:    "Jerry (Gerald) Sedgewick" <[hidden email]>
>>>
>>>
>>>
>>>
>>>> Subject: Re: Preparing figures for publication --PPI vs DPI
>>>
>>>
>>>
>>>
>>>>
>>>
>>>
>>>
>>>
>>>> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.01 Transitional//EN">
>>>
>>>
>>>
>>>
>>>> <html>
>>>
>>>
>>>
>>>
>>>> <head>
>>>
>>>
>>>
>>>
>>>> <meta content="text/html; charset=windows-1252"
>>>
>>>
>>>
>>>
>>>> http-equiv="Content-Type">
>>>
>>>
>>>
>>>
>>>> </head>
>>>
>>>
>>>
>>>
>>>> <body bgcolor="#ffffff" text="#000000">
>>>
>>>
>>>
>>>
>>>> Hi All,<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> maybe you might like to turn off HTML text formatting in your email
>> client when
>>>
>>>
>>>
>>>
>>> you post to the lists
>>>
>>>
>>>
>>>
>>> in this case its not so distracting but sometimes emails to this an
>> other list
>>>
>>>
>>>
>>>
>>> as unreadable in digest mode
>>>
>>>
>>>
>>>
>>> due to the thousands of html tags.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> I'm a little late in addressing this issue, but the PPI/DPI part of
>>>
>>>
>>>
>>>
>>>> this conversation is misleading.  What I mean to say is that the
>> real
>>>
>>>
>>>
>>>
>>>> issue may not be resolution, but reproduction.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> A very good point.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Images that are sent to
>>>
>>>
>>>
>>>
>>>> publishers with the full dynamic range of 0 - 255 pixel values may
>>>
>>>
>>>
>>>
>>>> likely be reproduced with no details at the bright end (240 - 255),
>> and
>>>
>>>
>>>
>>>
>>>> the dark end (0 - 20).  Printing presses cannot reproduce detail
>> within
>>>
>>>
>>>
>>>
>>>> these tonal ranges because of limitations with dropping ink on paper
>>>
>>>
>>>
>>>
>>>> without A) having the drop not stick when the tonal values are
>> bright
>>>
>>>
>>>
>>>
>>>> and B) having the inks blend into each other through capillary
>> action
>>>
>>>
>>>
>>>
>>>> at the darkest values.  This phenomenon gets worse when the paper is
>>>
>>>
>>>
>>>
>>>> lower quality (e.g., "Science").<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> So what we are saying is maybe summarised as follows:
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Small thumbnail images in print can only every be just that - pointers
>> to go and
>>>
>>>
>>>
>>>
>>> look at the real digital image on some online source.
>>>
>>>
>>>
>>>
>>> No one can hope to reach quantitative conclusions from a small print
>> image.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> We can probably consign print images on paper to the heap of old
>> technologies,
>>>
>>>
>>>
>>>
>>> which are no longer relevant to the work we do.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Images in PDF files "could" be useful, but not if they are lossy
>> compressed and
>>>
>>>
>>>
>>>
>>> missing meta data, which they usually are.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Color reproduction is generally worse because primary and secondary
>>>
>>>
>>>
>>>
>>>> colors are used to show experimental evidence.  These colors are not
>>>
>>>
>>>
>>>
>>>> always within the range of printing presses, and so these tend to
>> print
>>>
>>>
>>>
>>>
>>>> blobs sans detail when color choices are not appropriate for
>> publishing
>>>
>>>
>>>
>>>
>>>> onto paper.<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> To make matters worse....
>>>
>>>
>>>
>>>
>>> fors most peoples eyes, blue is much fainter than red and green, with
>> green
>>>
>>>
>>>
>>>
>>> often being precieved as brightest.
>>>
>>>
>>>
>>>
>>> So forget even semi  quantitative comparisons of intensity there.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> In any case we dont print in red green and blue, so these are silly
>> choices for
>>>
>>>
>>>
>>>
>>> a print version of figures.
>>>
>>>
>>>
>>>
>>> The print colour space would be better, Magenta, yellow and cyan....
>> but still
>>>
>>>
>>>
>>>
>>> for our quantitative purposed not good.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Screen (LCD/CRT) colours are more similar to out eyes trichromatic
>> response.
>>>
>>>
>>>
>>>
>>> That why they are red green and blue... but the blue looks darker then
>> green
>>>
>>>
>>>
>>>
>>> problem still persists.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> More often than not, the issue of whether or not details can be
>>>
>>>
>>>
>>>
>>>> resolved by eye on a printed page is not that of how many pixels
>> exist
>>>
>>>
>>>
>>>
>>>> in the submitted image, but in how effectively tones and colors were
>>>
>>>
>>>
>>>
>>>> fitted to the printing press.  This is especially true if the pdf
>> image
>>>
>>>
>>>
>>>
>>>> at non-zoomed, computer screen resolution reveals desired details,
>> but
>>>
>>>
>>>
>>>
>>>> the printed page does not:  it is more likely that a 133 line per
>> inch
>>>
>>>
>>>
>>>
>>>> screened image appearing in publication has more resolution than the
>>>
>>>
>>>
>>>
>>>> computer screen (often figured at an average of 90 pixels per inch,
>>>
>>>
>>>
>>>
>>>> with 72 pixels being the "old" standard).  The color and tonal range
>> of
>>>
>>>
>>>
>>>
>>>> reproduction of a computer screen is greater than on a printing
>> press.<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Indeed it is, and so is a better visualisation tool...
>>>
>>>
>>>
>>>
>>> but to get the most out of it, we also need to be smart.
>>>
>>>
>>>
>>>
>>> We cant compare the brightness of blue and green due to our
>> physiology,
>>>
>>>
>>>
>>>
>>> even if the screen is properly calibrated (usually not the case).
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> So, we should not ry to do that on screen or in print.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Our eyes are much better at discriminating greyscale brightness scale,
>>
>>>
>>>
>>>
>>>
>>> but actually we are sill pretty bad at that.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> see this example of where we are easily fooled when trying to compare
>> grey scale
>>>
>>>
>>>
>>>
>>> brightness.
>>>
>>>
>>>
>>>
>>> http://web.mit.edu/persci/people/adelson/checkershadow_illusion.html
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> The situation with colouyr is more complicated, and also beset with
>> pitfalle
>>>
>>>
>>>
>>>
>>> due to the way our optical system works... it tries to find contrast
>> in any
>>>
>>>
>>>
>>>
>>> scene,
>>>
>>>
>>>
>>>
>>> and even generates contrast or colour differences that are not there
>> (but gave
>>>
>>>
>>>
>>>
>>> us an evolutionary advantage in picking fruit)
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> See the spiral image at the bottom f this colocalisation tutorial,
>>>
>>>
>>>
>>>
>>> which shows us that we should be careful when interpreting colour
>> merge multi
>>>
>>>
>>>
>>>
>>> channel images.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> you can draw the spiral pattern yourself in imageJ with the marco
>> script there,
>>>
>>>
>>>
>>>
>>> and prove to yourself that your eyes lie to you.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Greyscale images can be made interpretable in a semi quantitative
>> manner by
>>>
>>>
>>>
>>>
>>> using for instance the fire colour look up table.
>>>
>>>
>>>
>>>
>>> Each bright colour represents some intensity value, so its easier to
>> compare
>>>
>>>
>>>
>>>
>>> intensities over and between images.
>>>
>>>
>>>
>>>
>>> Physicists and chemists do this all the time, and think we are mad for
>> showing
>>>
>>>
>>>
>>>
>>> DAPI staining  in black to Blue - because "thats what it looks like"
>>>
>>>
>>>
>>>
>>> They are right , we are mad to do that.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> Like others before this email, I believe that it is best that
>>>
>>>
>>>
>>>
>>>> scientists take the task of reproduction as much in their own hands
>> as
>>>
>>>
>>>
>>>
>>>> possible so that the outcome can be controlled.  The image is a
>>>
>>>
>>>
>>>
>>>> reproduction of what was once under a microscope, and it behooves
>> the
>>>
>>>
>>>
>>>
>>>> researcher to make that appear as close to the original
>> representation
>>>
>>>
>>>
>>>
>>>> as possible.  <br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> I'm not sure that this is ever going to be a helpful way to approach
>> the
>>>
>>>
>>>
>>>
>>> problem.
>>>
>>>
>>>
>>>
>>> No matter how carefully you set out your images and send them to the
>> publisher,
>>>
>>>
>>>
>>>
>>> you have no control and what they do with them next. None.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> The only way around this is to have the images in the paper act only
>> as
>>>
>>>
>>>
>>>
>>> thumbnails
>>>
>>>
>>>
>>>
>>> which point to the original image data file(s) on an online
>> repository,
>>>
>>>
>>>
>>>
>>> like the JCB image viewer or our wished for public biological image
>> database...
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> An image is not the sample, it always contain much less info that the
>> sample
>>>
>>>
>>>
>>>
>>> did.
>>>
>>>
>>>
>>>
>>> The trick is to keep the useful info as it passes through the scope
>> lenses,
>>>
>>>
>>>
>>>
>>> the detector, and the computer you your brain/eyes.
>>>
>>>
>>>
>>>
>>> I agree, an image is a representation of the info that made it through
>> the
>>>
>>>
>>>
>>>
>>> microscope to you.
>>>
>>>
>>>
>>>
>>> Trying to represent it "faithfully" and as  it "truly looks" are both
>> missing
>>>
>>>
>>>
>>>
>>> the mark.
>>>
>>>
>>>
>>>
>>> The image is usually degraded by the blur of the Point spread function
>> / OTF
>>>
>>>
>>>
>>>
>>> and by various sources of noise.
>>>
>>>
>>>
>>>
>>> Thus, the image is an artifact in of itself. What we want to know
>> about is the
>>>
>>>
>>>
>>>
>>> sample.
>>>
>>>
>>>
>>>
>>> The images contains info from the sample in a degraded  and incomplete
>> from.
>>>
>>>
>>>
>>>
>>> We have to work around that, and not pretend that the image fully
>> represents the
>>>
>>>
>>>
>>>
>>> sample.
>>>
>>>
>>>
>>>
>>> It does not.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> As far as images being data points, this also is true, and these are
>> to
>>>
>>>
>>>
>>>
>>>> remain unaltered (unless flatfield correcting, background
>> subtracting,
>>>
>>>
>>>
>>>
>>>> etc) for measuring.  A faithful representation of that image when
>>>
>>>
>>>
>>>
>>>> reproduced is another matter altogether.<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Indeed it is, and i think there is even no such thing as a faithful
>>>
>>>
>>>
>>>
>>> representation of the Sample as an image,
>>>
>>>
>>>
>>>
>>> so the faithful representation of the image is also then something to
>> think
>>>
>>>
>>>
>>>
>>> about.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> I think what matters is How you represent the image data....
>>>
>>>
>>>
>>>
>>> in some cases an illustration like Hooke's drawing of cells might even
>> be better
>>>
>>>
>>>
>>>
>>> than a digital representation,
>>>
>>>
>>>
>>>
>>> if you want to get a certain message across to the reader. Original
>> digital
>>>
>>>
>>>
>>>
>>> image data available too of course online.
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> cheers
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Dan
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Cheers,<br>
>>>
>>>
>>>
>>>
>>>> <br>
>>>
>>>
>>>
>>>
>>>> Jerry Sedgewick<br>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> Dr. Daniel James White BSc. (Hons.) PhD
>>>
>>>
>>>
>>>
>>> Senior Microscopist / Image Visualisation, Processing and Analysis
>>>
>>>
>>>
>>>
>>> Light Microscopy and Image Processing Facilities
>>>
>>>
>>>
>>>
>>> Max Planck Institute of Molecular Cell Biology and Genetics
>>>
>>>
>>>
>>>
>>> Pfotenhauerstrasse 108
>>>
>>>
>>>
>>>
>>> 01307 DRESDEN
>>>
>>>
>>>
>>>
>>> Germany
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> [phone] (German Mobile)
>>>
>>>
>>>
>>>
>>> [phone] (Work phone at MPI-CBG)
>>>
>>>
>>>
>>>
>>> [phone] (Fax MPI-CBG LMF)
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>>
>>> http://www.bioimagexd.net   BioImageXD
>>>
>>>
>>>
>>>
>>> http://pacific.mpi-cbg.de       Fiji -  is just ImageJ (Batteries
>> Included)
>>>
>>>
>>>
>>>
>>> http://www.chalkie.org.uk       Dan's Homepages
>>>
>>>
>>>
>>>
>>> https://ifn.mpi-cbg.de          Dresden Imaging Facility Network
>>>
>>>
>>>
>>>
>>> dan (at) chalkie.org.uk
>>>
>>>
>>>
>>>
>>> ( white (at) mpi-cbg.de )
>>>
>>>
>>>
>>>
>>>
>>> ---------------------------------------------------------------------
>>> SECURITY/CONFIDENTIALITY WARNING:
>>> This message and any attachments are intended solely for the
>> individual or entity to which they are addressed. This communication may
>> contain information that is privileged, confidential, or exempt from
>> disclosure under applicable law (e.g., personal health information,
>> research data, financial information). Because this e-mail has been sent
>> without encryption, individuals other than the intended recipient may be
>> able to view the information, forward it to others or tamper with the
>> information without the knowledge or consent of the sender. If you are
>> not the intended recipient, or the employee or person responsible for
>> delivering the message to the intended recipient, any dissemination,
>> distribution or copying of the communication is strictly prohibited. If
>> you received the communication in error, please notify the sender
>> immediately by replying to this message and deleting the message and any
>> accompanying files from your system. If, due to the security risks, you
>> do not wish to receive further communications via e-mail, please reply
>> to this message and inform the sender that you do not wish to receive
>> further e-mail from the sender.
>>> ---------------------------------------------------------------------
>>
>> Dr. David Knecht
>> Department of Molecular and Cell Biology
>> Co-head Flow Cytometry and Confocal Microscopy Facility
>> U-3125
>> 91 N. Eagleville Rd.
>> University of Connecticut
>> Storrs, CT 06269
>> 860-486-2200
>> 860-486-4331 (fax)
>

--
Dr. Dmitry Sokolov
Institute of Fundamental Sciences
Massey University, Palmerston North
New Zealand