Re: digital imaging standard - Summary and further questions

[Z.J. Zhang]

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To join, leave or search the confocal microscopy listserv, go to:
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Thank you all for your response to my early post "digital imaging standard".

Although the question was very broad and vague,  I received many responses that are very helpful! Many of the responses point to guidelines, rather than "standards", but they are important for the discussion.

Here is a long summary and I hope this can stimulate more discussion (and research?) to this exciting (and confusing) topic. Two more related questions are posted at the end.


SUMMARY:
The standards for digital imaging (especially for microscopy imaging) could include, at least, the following aspects:

Acquisition/Capturing (including digitization of non-digital images?);

Storage;

Processing/manipulation/analysis/

Digital imaging ethics;

Image output



1.      Acquisition/capturing



1a: the health of the microscope:


---------------

John Oreopoulos [[hidden email]]



There are several different standards for measuring spatial resolution: sub-diffraction sized fluorescent beads, mirrors, etc. Fluorescent beads can be purchased from many different companies, but there is no agreed upon standard size, dye spectra, or anything like that. Some people have used flow cytometry beads before, and I think the quality control is a bit tighter on those.



Molecular Probes also sells a fluorescent bead kit with beads that step up their fluorescent intensity in known amounts (no commercial interest):



http://probes.invitrogen.com/media/pis/mp07219.pdf



There are also standard resolution targets for brightfield and reflected imaging. Here's an example from Edmund Optics (no commercial interest):



http://www.edmundoptics.com/products/displayproduct.cfm?productid=1790



Pawley's confocal handbook says that you can coat these with a fluorescent dye if you want to use them for fluorescence.



Unfortunately, there is no intensity standard or "standard candle" for microscopy, although as you may have seen in some recent posts, some people suggest using the transmitted light source of the microscope and a power meter. I think uranyl glass has been talked about several times as well. If someone does have a good intensity standard, I'd really like to know about it as well.


-----------------

Rich Cole [[hidden email]] and Anda Cornea [[hidden email]]



ABRF (The Association of Biomolecular Resource Facilities) studies

http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm



Stack, R., Bayles, C., Girard, A., Martin, K., Opansky, C., Schulz, K., and Cole, R. (2011) Quality Assurance Testing for Modern Optical Imaging Systems. Microscopy & Microanalysis 17(4):598-606.



They use Chroma fluorescent plastic slides, sub-resolution beads, orange/orange spectral separation beads and reflection off gold coated slide.  All good, none perfect.



For the health of my SP5 AOBS, I run a daily a 3 minute test that involves x-z-lambda, (occasionally x-z-lambda-t) scanning in reflection mode off a plain glass slide with all lasers on, same parameters of course.  It gives a lot of information and it makes it relatively easy to detect problems and pinpoint their sources.



1b: Image acquisition/capturing


JOEL B. SHEFFIELD [[hidden email]]



There may not be formal standards, but I would certainly think that there are principles that should guide digital microscopy.

1.  The pixel density should correspond, at a minimum, to the relevant detail in the original source.



2.  The bit depth should include all levels of intensity in the original --i.e. not saturated, and not clipped.



3.  For microscopy, images should include a magnification standard.



------------------------------

Nestor [[hidden email]<mailto:[hidden email]>]



JPEG formating  CHANGES the data. What is even worse if you take a JPEG image and store it a second time CHANGES are made to the last set of  CHANGES.  This corrupts the  integrity of the scientific  data.



Do a simple test.  Take an image store it in TIFF.

Then store it as JPEG, then open the JPEG and compare the results pixel by pixel. You no longer have the same information.  If you then store the JPEG again the compression routine reformat the data yet again.



You should always, repeat always, store your original data in a lossless format. RAW, TIFF are two of the examples.  You can just JPEG to post on the WWW or print posters, but never when any quantitative work is being done from that data set.





2.      Storage and image analysis



-------------------

Geoff McAuliffe [[hidden email]]



Do not use a "lossy" compression, ie. JPEG. Use TIFF to save your images. Journals will publish guidelines in the "Instructions to

Authors" section as to what, if any, modifications may be allowed. Always save the original, unaltered image file and a backup.


-----------------------
Jennifer Hill [[hidden email]]


In my experiences, most researchers use a tiff file format for publications and lossless compression jpegs for rough review. As far as a standard goes, there should be no manipulation in photoshop or similar programs that alters the original specimen from its original state. Adjusting brightness/contrast is usually ok; and depending on the purpose/ final output of the image the background can be cleaned of dust/noise. I'd be happy to answer any more of your questions in regards to imaging.



Ps. I agree with the other individual who posted; a scale of magnification should be included with the image.



--------------------
Jim Quinn [[hidden email]]



There are many ASTM standards for microscopy, some are digital, such as E1122 for JK inclusion rating by automated image analysis.  E2765 for image capture and storage.  The E4 subcommittee chair is John Friel. He can likely point you in the correct direction.


---------------------
Donnelly, Tom [[hidden email]]

As far as standards for mfg. there really is nothing out there.  There is an industry group, OPIA, that meets one or two times per year to discuss "standards".  Unfortunately each of the "Big Four" are all for standards for data files, operational communication, etc. as long as the standards conform to their individual formats.



The smaller companies which create software and third party hardware, as a group would support across the board standardization because every time Zeiss, Leica, Nikon, or Olympus change a hardware design or a software data input/output the third party vendors have to redesign their products.



This conflict will probably prevent true standards for a long time.



The best chance for data standards, at least, is the Open Microscopy Environment, project. http://www.openmicroscopy.org/site   This project allows imaging facilities to utilize a single data format for analysis and storage.


--------------------------------
Dmitry Sokolov [[hidden email]]


MIAWiki, Wiki for Microscopy and Image Analysis, is a collaboration tool where primary information like papers, patents, etc. can be cited, analysed, published and shared for free. In many cases R&D is based on the best practices and protocols rather than standards.



Please discover capabilities of MIAWiki of real time accumulation and finding knowledge on your current demand.

http://confocal-manawatu.pbworks.com/w/page/16346911/FrontPage



-----------------------
Stan Schwartz [[hidden email]]

The International Organization for Standardization  covers this.
http://www.iso.org/iso/search.htm?qt=digital+image&sort=rel&type=simple&published=on

Along with many standards for Microscopy.
http://www.iso.org/iso/search.htm?qt=microscopy&sort=rel&type=simple&published=on
NOTE: The documents can be only access on a pay basis. There is not even a summary available.


3.      Digital imaging ethics


-------------------------

John Oreopoulos [[hidden email]]



there is a fantastic article by Douglas Cromey that thoroughly discusses the topic:



Cromey, D., Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images. Science and Engineering Ethics, 2010.


--------------------------
Mike Bode [[hidden email]]

I don't think there is a standard as to what file format to use or what image processing software to use, but there are ethical guidelines as to what can be done and what should not be done, and how the image manipulations should be documented. One such article you can find here:



http://swehsc.pharmacy.arizona.edu/exppath/micro/digimage_ethics.php



MSA also has a statement on ethical image processing.



http://www.microscopy.org/resources/digital_imaging.cfm



The gist of both is that you need to stay as true as possible to the original image and pixel data (no compression), and that anything that could lead to artifacts needs to be documented, and, of course, that whatever you do to the image is documented in such a fashion that it can be repeated (as all scientific data should).



--------------------

4. Image output



Are we still reined by the mercy of the publishers? Often I see publishers require a resolution of 1200ppi, but no mention the image size. It ends up a "high resolution" image but the print size is 0.01 inches. Any thought on that?



Further questions:

1.       Remember in the "old" days, that all films have a speed, ISO 100, 200 etc.  Nowadays, do we just use the "gain" to control the speed and "grain size" on the digital camera? Is there a "standard"?

2.       Quantitative analysis is becoming a norm of digital imaging. Does it always require 16bit images, rather 8bit? Did anyone compare the same sample using 8bit or 16bit, and see a significant difference?





Zhaojie

Zhaojie Zhang, Ph. D.
Director, Jenkins Microscopy Facility
University of Wyoming
Laramie, WY 82071
PHONE: [phone]
FAX: [phone]

[Dmitry Sokolov]

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

Dear All,

thank you for your contribution.
Here is the permalink on the topic at MIAWiki for your future reference:
http://confocal-manawatu.pbworks.com/w/page/47001491/Digital%20Imaging%20Standards
Your answers have been assembled by Zhaojie and ready for further
review, analysis and publishing in more generalized form for quicker
access to particular subtopics in future.

Thanks again,
Dmitry

Z.J. Zhang wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thank you all for your response to my early post "digital imaging standard".
>
> Although the question was very broad and vague,  I received many responses that are very helpful! Many of the responses point to guidelines, rather than "standards", but they are important for the discussion.
>
> Here is a long summary and I hope this can stimulate more discussion (and research?) to this exciting (and confusing) topic. Two more related questions are posted at the end.
>
>
> SUMMARY:
> The standards for digital imaging (especially for microscopy imaging) could include, at least, the following aspects:
>
> Acquisition/Capturing (including digitization of non-digital images?);
>
> Storage;
>
> Processing/manipulation/analysis/
>
> Digital imaging ethics;
>
> Image output
>
>
>
> 1.      Acquisition/capturing
>
>
>
> 1a: the health of the microscope:
>
>
> ---------------
>
> John Oreopoulos [[hidden email]]
>
>
>
> There are several different standards for measuring spatial resolution: sub-diffraction sized fluorescent beads, mirrors, etc. Fluorescent beads can be purchased from many different companies, but there is no agreed upon standard size, dye spectra, or anything like that. Some people have used flow cytometry beads before, and I think the quality control is a bit tighter on those.
>
>
>
> Molecular Probes also sells a fluorescent bead kit with beads that step up their fluorescent intensity in known amounts (no commercial interest):
>
>
>
> http://probes.invitrogen.com/media/pis/mp07219.pdf
>
>
>
> There are also standard resolution targets for brightfield and reflected imaging. Here's an example from Edmund Optics (no commercial interest):
>
>
>
> http://www.edmundoptics.com/products/displayproduct.cfm?productid=1790
>
>
>
> Pawley's confocal handbook says that you can coat these with a fluorescent dye if you want to use them for fluorescence.
>
>
>
> Unfortunately, there is no intensity standard or "standard candle" for microscopy, although as you may have seen in some recent posts, some people suggest using the transmitted light source of the microscope and a power meter. I think uranyl glass has been talked about several times as well. If someone does have a good intensity standard, I'd really like to know about it as well.
>
>
> -----------------
>
> Rich Cole [[hidden email]] and Anda Cornea [[hidden email]]
>
>
>
> ABRF (The Association of Biomolecular Resource Facilities) studies
>
> http://www.abrf.org/index.cfm/group.show/LightMicroscopyResearchGroup.54.htm
>
>
>
> Stack, R., Bayles, C., Girard, A., Martin, K., Opansky, C., Schulz, K., and Cole, R. (2011) Quality Assurance Testing for Modern Optical Imaging Systems. Microscopy & Microanalysis 17(4):598-606.
>
>
>
> They use Chroma fluorescent plastic slides, sub-resolution beads, orange/orange spectral separation beads and reflection off gold coated slide.  All good, none perfect.
>
>
>
> For the health of my SP5 AOBS, I run a daily a 3 minute test that involves x-z-lambda, (occasionally x-z-lambda-t) scanning in reflection mode off a plain glass slide with all lasers on, same parameters of course.  It gives a lot of information and it makes it relatively easy to detect problems and pinpoint their sources.
>
>
>
> 1b: Image acquisition/capturing
>
>
> JOEL B. SHEFFIELD [[hidden email]]
>
>
>
> There may not be formal standards, but I would certainly think that there are principles that should guide digital microscopy.
>
> 1.  The pixel density should correspond, at a minimum, to the relevant detail in the original source.
>
>
>
> 2.  The bit depth should include all levels of intensity in the original --i.e. not saturated, and not clipped.
>
>
>
> 3.  For microscopy, images should include a magnification standard.
>
>
>
> ------------------------------
>
> Nestor [[hidden email]<mailto:[hidden email]>]
>
>
>
> JPEG formating  CHANGES the data. What is even worse if you take a JPEG image and store it a second time CHANGES are made to the last set of  CHANGES.  This corrupts the  integrity of the scientific  data.
>
>
>
> Do a simple test.  Take an image store it in TIFF.
>
> Then store it as JPEG, then open the JPEG and compare the results pixel by pixel. You no longer have the same information.  If you then store the JPEG again the compression routine reformat the data yet again.
>
>
>
> You should always, repeat always, store your original data in a lossless format. RAW, TIFF are two of the examples.  You can just JPEG to post on the WWW or print posters, but never when any quantitative work is being done from that data set.
>
>
>
>
>
> 2.      Storage and image analysis
>
>
>
> -------------------
>
> Geoff McAuliffe [[hidden email]]
>
>
>
> Do not use a "lossy" compression, ie. JPEG. Use TIFF to save your images. Journals will publish guidelines in the "Instructions to
>
> Authors" section as to what, if any, modifications may be allowed. Always save the original, unaltered image file and a backup.
>
>
> -----------------------
> Jennifer Hill [[hidden email]]
>
>
> In my experiences, most researchers use a tiff file format for publications and lossless compression jpegs for rough review. As far as a standard goes, there should be no manipulation in photoshop or similar programs that alters the original specimen from its original state. Adjusting brightness/contrast is usually ok; and depending on the purpose/ final output of the image the background can be cleaned of dust/noise. I'd be happy to answer any more of your questions in regards to imaging.
>
>
>
> Ps. I agree with the other individual who posted; a scale of magnification should be included with the image.
>
>
>
> --------------------
> Jim Quinn [[hidden email]]
>
>
>
> There are many ASTM standards for microscopy, some are digital, such as E1122 for JK inclusion rating by automated image analysis.  E2765 for image capture and storage.  The E4 subcommittee chair is John Friel. He can likely point you in the correct direction.
>
>
> ---------------------
> Donnelly, Tom [[hidden email]]
>
> As far as standards for mfg. there really is nothing out there.  There is an industry group, OPIA, that meets one or two times per year to discuss "standards".  Unfortunately each of the "Big Four" are all for standards for data files, operational communication, etc. as long as the standards conform to their individual formats.
>
>
>
> The smaller companies which create software and third party hardware, as a group would support across the board standardization because every time Zeiss, Leica, Nikon, or Olympus change a hardware design or a software data input/output the third party vendors have to redesign their products.
>
>
>
> This conflict will probably prevent true standards for a long time.
>
>
>
> The best chance for data standards, at least, is the Open Microscopy Environment, project. http://www.openmicroscopy.org/site   This project allows imaging facilities to utilize a single data format for analysis and storage.
>
>
> --------------------------------
> Dmitry Sokolov [[hidden email]]
>
>
> MIAWiki, Wiki for Microscopy and Image Analysis, is a collaboration tool where primary information like papers, patents, etc. can be cited, analysed, published and shared for free. In many cases R&D is based on the best practices and protocols rather than standards.
>
>
>
> Please discover capabilities of MIAWiki of real time accumulation and finding knowledge on your current demand.
>
> http://confocal-manawatu.pbworks.com/w/page/16346911/FrontPage
>
>
>
> -----------------------
> Stan Schwartz [[hidden email]]
>
> The International Organization for Standardization  covers this.
> http://www.iso.org/iso/search.htm?qt=digital+image&sort=rel&type=simple&published=on
>
> Along with many standards for Microscopy.
> http://www.iso.org/iso/search.htm?qt=microscopy&sort=rel&type=simple&published=on
> NOTE: The documents can be only access on a pay basis. There is not even a summary available.
>
>
> 3.      Digital imaging ethics
>
>
> -------------------------
>
> John Oreopoulos [[hidden email]]
>
>
>
> there is a fantastic article by Douglas Cromey that thoroughly discusses the topic:
>
>
>
> Cromey, D., Avoiding Twisted Pixels: Ethical Guidelines for the Appropriate Use and Manipulation of Scientific Digital Images. Science and Engineering Ethics, 2010.
>
>
> --------------------------
> Mike Bode [[hidden email]]
>
> I don't think there is a standard as to what file format to use or what image processing software to use, but there are ethical guidelines as to what can be done and what should not be done, and how the image manipulations should be documented. One such article you can find here:
>
>
>
> http://swehsc.pharmacy.arizona.edu/exppath/micro/digimage_ethics.php
>
>
>
> MSA also has a statement on ethical image processing.
>
>
>
> http://www.microscopy.org/resources/digital_imaging.cfm
>
>
>
> The gist of both is that you need to stay as true as possible to the original image and pixel data (no compression), and that anything that could lead to artifacts needs to be documented, and, of course, that whatever you do to the image is documented in such a fashion that it can be repeated (as all scientific data should).
>
>
>
> --------------------
>
> 4. Image output
>
>
>
> Are we still reined by the mercy of the publishers? Often I see publishers require a resolution of 1200ppi, but no mention the image size. It ends up a "high resolution" image but the print size is 0.01 inches. Any thought on that?
>
>
>
> Further questions:
>
> 1.       Remember in the "old" days, that all films have a speed, ISO 100, 200 etc.  Nowadays, do we just use the "gain" to control the speed and "grain size" on the digital camera? Is there a "standard"?
>
> 2.       Quantitative analysis is becoming a norm of digital imaging. Does it always require 16bit images, rather 8bit? Did anyone compare the same sample using 8bit or 16bit, and see a significant difference?
>
>
>
>
>
> Zhaojie
>
> Zhaojie Zhang, Ph. D.
> Director, Jenkins Microscopy Facility
> University of Wyoming
> Laramie, WY 82071
> PHONE: [phone]
> FAX: [phone]
>
>
>  

[Straatman, Kees (Dr.)]

Dear list members,

I would like to announce the next ImageJ workshops: 'Introduction to ImageJ/FIJI' on Monday 19 December and 'Writing macros in ImageJ' on Tuesday 20 December. The workshops in July were absolutely oversubscribed and I got many request for a repeat. Maximum 24 participants.

Please visit the website: http://www.le.ac.uk/biochem/microscopy/ImageJ2011b.html

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology

http://www.le.ac.uk/biochem/microscopy/home.html

[Asson-Batres, Mary Ann]

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

Thanks for the information.  I do wish this could be held virtually - the UK is out of reach for me, but the workshops sound excellent.



I wonder if there are other IMAGE J workshops that are offered online?



Mary Ann



Mary Ann Asson-Batres
Professor, Department of Biological Sciences
Tennessee State University

[hidden email]<mailto:[hidden email]>
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Straatman, Kees R. (Dr.) [[hidden email]]
Sent: Tuesday, October 18, 2011 7:54 AM
To: [hidden email]
Subject: ImageJ/Fiji workshops 19 and 20 December

Dear list members,

I would like to announce the next ImageJ workshops: 'Introduction to ImageJ/FIJI' on Monday 19 December and 'Writing macros in ImageJ' on Tuesday 20 December. The workshops in July were absolutely oversubscribed and I got many request for a repeat. Maximum 24 participants.

Please visit the website: http://www.le.ac.uk/biochem/microscopy/ImageJ2011b.html

Best wishes

Kees


Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology

http://www.le.ac.uk/biochem/microscopy/home.html