Deconvolution of Transmission Images

[Urs Utzinger]

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Most deconvolution microscopy is concerned about fluorescent samples.
However it is possible to processes laser scanning transmission images in a similar
fashion. Those images are usually obtained by measuring the intensity of the
transmitted laser beam in a non-descanning fashion. Best contrast is obtained by
stopping down the condenser because fluctuations in the refractive index of the
sample make it appear similar to phase contrast microscopy.

Such transmission data is not optically sectioned like confocal or 2P images. There
are several publications about using deconvolution approaches for transmission
electron microscopy.

I would like to ask if anyone is aware of publications or work conducted on
"deconvolving" optical transmission or phase contrast data stacks in order to
improve Z resolution.

Urs Utzinger
University of Arizona

[George McNamara]

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Hi Urs,

Chapter 24 on "Blind deconvolution" of Pawley 2006 Handbook by Holmes,
Biggs, and Abu-Tarif. You can check the AutoQuant product line at Media
Cybernetics to see if brightfield deconvolution is in there.

As for optical sectioning in transmitted light, Shinya Inoue in Video
Microscopy (1st ed - I don't recall Spring & Inoue in this detail)
showed very nice optical sectioning of a preparation using phase contrast.

I believe IATIA (now ultimacapital.net/iatiaimaging ) QPm Z-series have
Z-resolved maps. Search for "iatia qpm" on the internet. For starters, see

http://aups.org.au/Proceedings/34/121-127/121-127.pdf
/www.focusonmicroscopy.org/2004/abstracts/056_Xiang.pdf
www.ultimacapital.net/*iatia*imaging
/
QPm is inside the GE InCell series HCS instruments (now handled by
Applied Precision). I have encouraged GE/API to take advantage of their
license to
(1) give InCell users the QPm map data - instead of just providing a
user interface to spew out the dumbed down phase contrast like image
(complete with halo!).
(2) integrate QPm into the DeltaVision deconvolution and the OMX 3D-SIM
nanoscope lines (every OMX ships with the deconvolution software - I
have not mentioned that QPm of structured illumination source data
should have even better optical sectioning since their marketing dept is
struggling with the idea of QPm as yet another quantitative imaging mode
... I did mention that an InCell with 3D-SIM and their newly tweaked PCO
sCMOS could be even more fun - single molecule counting - than the
somewhat clever linescan confocal mode in the InCell 6000).

Enjoy,


George



On 3/20/2012 10:02 AM, Urs Utzinger wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Most deconvolution microscopy is concerned about fluorescent samples.
> However it is possible to processes laser scanning transmission images in a similar
> fashion. Those images are usually obtained by measuring the intensity of the
> transmitted laser beam in a non-descanning fashion. Best contrast is obtained by
> stopping down the condenser because fluctuations in the refractive index of the
> sample make it appear similar to phase contrast microscopy.
>
> Such transmission data is not optically sectioned like confocal or 2P images. There
> are several publications about using deconvolution approaches for transmission
> electron microscopy.
>
> I would like to ask if anyone is aware of publications or work conducted on
> "deconvolving" optical transmission or phase contrast data stacks in order to
> improve Z resolution.
>
> Urs Utzinger
> University of Arizona
>
>    

--


George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility (AICF)
University of Miami, Miller School of Medicine

http://www.sylvester.org/AICF   (AICF home page)
PubSpectra data (XLSX file inside)
    http://www.sylvester.org/documents/PubSpectra.zip (download 2000+ spectra)
    http://works.bepress.com/gmcnamara/
PubSpectra / UA Graphing Site
     http://www.mcb.arizona.edu/ipc/fret/index.html   (Carl Boswell, now retired)
New UA Spectra Database Site
     http://www.spectra.arizona.edu/                  (Urs Utzinger)
UMiami Scholarly Repository "selected works"
     http://works.bepress.com/gmcnamara
Care to link?
     http://www.linkedin.com/in/georgemcnamara


Ready for imaging in 2012? Check out:


Miami 2012 Winter Symposium: Nanotechnology in Biomedicine
February 26-29, 2012, Miami, FL
Nature Publishing Group / University of Miami / Scripps Florida
http://www.nature.com/natureconferences/miami/mws2012/speakers.html

Association of Biomolecular Resource Facilities (ABRF)
International Symposium
March 17-20, 2012, Orlando, FL
http://conf.abrf.org/index.cfm

Biomedical Optics 2012 (OSA BIOMED) - Optical Society of America
April 29-May 2, 2012, Miami, FL
http://www.osa.org/meetings/topical_meetings/BIOMED/default.aspx



"Old soldiers never die, they just fade away." - Douglas Macarthur.
"Old antibodies die, please throw them away." - GM.

"Well of course you can't understand your data, you have too many controls" - Anna M. Wu, quoted in Andreas Markus Loening, Ph.D. dissertation, UCLA, 2006.
"If you do all the controls, you'll never publish." - GM.
"If you don't do the controls, you shouldn't publish." ... alternative: "If you don't do the controls, don't waste everyone's time in lab meeting." - GM.





 

[Renato A. Mortara]

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Dear all,

Having attended the first Pawley course in Vancouver I feel highly
embarassed to ask this, but I would really appreciate a clarification:

When estimating the highest zoom users should apply to their sample in order
to accommodate for the Nyquist theorem, I estimated the optimum pixel size
value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
the value is approxiametely 90 nm.

The doubt: if the image size is increased from 512x512 (having adjusted the
zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
(displayed by the system - Leica) the pixel size decreases 4 fold, to 22.5
nm. Since the resolution obviously did not change but only the image size,
what happens to Nyquist and the optimum pixel size at 2Kx2K ?

Many thanks !

Renato

Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara

[John Oreopoulos]

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Renato,

Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.

You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.

Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.

Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-04-11, at 7:22 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Having attended the first Pawley course in Vancouver I feel highly
> embarassed to ask this, but I would really appreciate a clarification:
>
> When estimating the highest zoom users should apply to their sample in order
> to accommodate for the Nyquist theorem, I estimated the optimum pixel size
> value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
> the value is approxiametely 90 nm.
>
> The doubt: if the image size is increased from 512x512 (having adjusted the
> zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
> (displayed by the system - Leica) the pixel size decreases 4 fold, to 22.5
> nm. Since the resolution obviously did not change but only the image size,
> what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara

[Guy Cox-2]

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

Just to add a bit to John's absolutely correct explanation.  

It's basically a question of interpretation.  You set up Nyquist imaging at 512x512 and then selected 2048x2048, expecting to get the same resolution but a 4 times bigger area.  Perfectly reasonable.  But the Leica software assumed you now wanted to get 2048x2048 pixels within the same chosen field of view.  Also a perfectly reasonable interpretation.  One might, in a perfect world, expect the software to ask you which interpretation you want, but if it doesn't it's pretty easy to fix.

                                              Guy


Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
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 John Oreopoulos
Sent: Wednesday, 11 April 2012 10:29 PM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Renato,

Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.

You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.

Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.

Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-04-11, at 7:22 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Having attended the first Pawley course in Vancouver I feel highly
> embarassed to ask this, but I would really appreciate a clarification:
>
> When estimating the highest zoom users should apply to their sample in order
> to accommodate for the Nyquist theorem, I estimated the optimum pixel size
> value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
> the value is approxiametely 90 nm.
>
> The doubt: if the image size is increased from 512x512 (having adjusted the
> zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
> (displayed by the system - Leica) the pixel size decreases 4 fold, to 22.5
> nm. Since the resolution obviously did not change but only the image size,
> what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara

[Lemasters, John J.]

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*****

Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.

John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury & Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Wednesday, April 11, 2012 8:29 AM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Renato,

Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.

You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.

Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.

Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-04-11, at 7:22 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Having attended the first Pawley course in Vancouver I feel highly
> embarassed to ask this, but I would really appreciate a clarification:
>
> When estimating the highest zoom users should apply to their sample in
> order to accommodate for the Nyquist theorem, I estimated the optimum
> pixel size value by dividing the lateral resolution (eg: 0.2 microns)
> by 2.3 so that the value is approxiametely 90 nm.
>
> The doubt: if the image size is increased from 512x512 (having
> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
> pixel size (displayed by the system - Leica) the pixel size decreases
> 4 fold, to 22.5 nm. Since the resolution obviously did not change but
> only the image size, what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara

[Monique Vasseur]

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

Hi John,

Indirectly, do you suggest the same for Z sampling if we are interested in 3D measurements?  Thanks

Monique Vasseur

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Lemasters, John J.
Envoyé : 11 avril 2012 09:34
À : [hidden email]
Objet : Re: Nyquist and Image size

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

Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.

John

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury & Regeneration Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Wednesday, April 11, 2012 8:29 AM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Renato,

Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.

You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.

Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.

Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-04-11, at 7:22 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> Having attended the first Pawley course in Vancouver I feel highly
> embarassed to ask this, but I would really appreciate a clarification:
>
> When estimating the highest zoom users should apply to their sample in
> order to accommodate for the Nyquist theorem, I estimated the optimum
> pixel size value by dividing the lateral resolution (eg: 0.2 microns)
> by 2.3 so that the value is approxiametely 90 nm.
>
> The doubt: if the image size is increased from 512x512 (having
> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
> pixel size (displayed by the system - Leica) the pixel size decreases
> 4 fold, to 22.5 nm. Since the resolution obviously did not change but
> only the image size, what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara

[Craig Brideau]

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

I've always thought of Zoom as my field-of-view control.  Many microscope
systems let you set a sub-region to scan, and depending on the software
this can be either zoom or crop.  Some software lets you select whether it
acts as either or when you readjust the size of the sub-region selection
box.

Craig


On Wed, Apr 11, 2012 at 7:05 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Just to add a bit to John's absolutely correct explanation.
>
> It's basically a question of interpretation.  You set up Nyquist imaging
> at 512x512 and then selected 2048x2048, expecting to get the same
> resolution but a 4 times bigger area.  Perfectly reasonable.  But the Leica
> software assumed you now wanted to get 2048x2048 pixels within the same
> chosen field of view.  Also a perfectly reasonable interpretation.  One
> might, in a perfect world, expect the software to ask you which
> interpretation you want, but if it doesn't it's pretty easy to fix.
>
>                                              Guy
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate,
> 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 John Oreopoulos
> Sent: Wednesday, 11 April 2012 10:29 PM
> To: [hidden email]
> Subject: Re: Nyquist and Image size
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Renato,
>
> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist
> sampling rate (ie: pixel dimensions) does not change since your objective
> lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm
> x 22.5 nm) means you are oversampling the image (and not gaining anything).
> Your image may look smoother but it contains no more information than the
> 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the
> same between 512x512 and 2Kx2K.
>
> You should decrease the galvometric mirror scan zoom setting to get back
> to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
> Effectively, you will be imaging (and properly sampling) a larger field of
> view then. I'm not familiar with the Leica laser scanning confocals so I'm
> not sure if it will allow you to do this. On other systems, like the
> Olympus FV300 for example, you can set your image pixel dimensions
> (256x256, 512x512, etc.) and your scan zoom independently.
>
> Just out of curiosity, why image 2K x 2K when you can't easily display
> that on a standard computer screen or present it in a published paper
> without downsizing? I rarely departed from 512x512 in my laser scanning
> days, except when I wanted to see a larger field of view.
>
> Cheers,
>
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear all,
> >
> > Having attended the first Pawley course in Vancouver I feel highly
> > embarassed to ask this, but I would really appreciate a clarification:
> >
> > When estimating the highest zoom users should apply to their sample in
> order
> > to accommodate for the Nyquist theorem, I estimated the optimum pixel
> size
> > value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
> > the value is approxiametely 90 nm.
> >
> > The doubt: if the image size is increased from 512x512 (having adjusted
> the
> > zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
> > (displayed by the system - Leica) the pixel size decreases 4 fold, to
> 22.5
> > nm. Since the resolution obviously did not change but only the image
> size,
> > what happens to Nyquist and the optimum pixel size at 2Kx2K ?
> >
> > Many thanks !
> >
> > Renato
> >
> > Renato A. Mortara
> > Parasitology Division
> > UNIFESP - Escola Paulista de Medicina
> > Rua Botucatu, 862, 6th floor
> > São Paulo, SP
> > 04023-062
> > Brazil
> > Phone: [phone]
> > Fax:     [phone]
> > email: [hidden email]
> > home page: www.ecb.epm.br/~ramortara
>

[David Baddeley]

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


The diagonal in z will be much 'straighter' (due to the fact that the voxels are elongated in z rather than being square), making the factor much closer to 1 (probably something like 1.1) so it can safely be ignored. When talking about slightly oversampling, 2.3 is already doing this - strict Nyquist is a factor of 2. It's also worth noting that you should probably use the theoretical resolution values (ie ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and not the observed PSF width, as these reflect the bandwidth of the system. I this tend to reccommend a blanket 70x70x200nm pixel size when using a high NA objective on fixed cells. In live cells, or other delicate samples you need to exercise a little more discretion - the artefacts introduced by slight undersampling are likely to be outweighed by other considerations.

My 2c,
David


------------------------------
On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi John,
>
>Indirectly, do you suggest the same for Z sampling if we are interested in 3D measurements?  Thanks
>
>Monique Vasseur
>
>-----Message d'origine-----
>De : Confocal Microscopy List [mailto:[hidden email]] De la part de Lemasters, John J.
>Envoyé : 11 avril 2012 09:34
>À : [hidden email]
>Objet : Re: Nyquist and Image size
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.
>
>John
>
>--
>John J. Lemasters, MD, PhD
>Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury & Regeneration Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
>DD504 Drug Discovery Building
>70 President Street, MSC 140
>Charleston, SC 29425
>
>Office: 843-876-2360
>Lab: 843-876-2354
>Fax: [phone]
>Email: [hidden email]
>http://academicdepartments.musc.edu/ccdir
>
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
>Sent: Wednesday, April 11, 2012 8:29 AM
>To: [hidden email]
>Subject: Re: Nyquist and Image size
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Renato,
>
>Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.
>
>You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>
>Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.
>
>Cheers,
>
>
>John Oreopoulos
>Research Assistant
>Spectral Applied Research
>Richmond Hill, Ontario
>Canada
>www.spectral.ca
>
>
>On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> Having attended the first Pawley course in Vancouver I feel highly
>> embarassed to ask this, but I would really appreciate a clarification:
>>
>> When estimating the highest zoom users should apply to their sample in
>> order to accommodate for the Nyquist theorem, I estimated the optimum
>> pixel size value by dividing the lateral resolution (eg: 0.2 microns)
>> by 2.3 so that the value is approxiametely 90 nm.
>>
>> The doubt: if the image size is increased from 512x512 (having
>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
>> pixel size (displayed by the system - Leica) the pixel size decreases
>> 4 fold, to 22.5 nm. Since the resolution obviously did not change but
>> only the image size, what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>>
>> Many thanks !
>>
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara

[George McNamara]

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

I agree with John Lemasters on XY:

    John: Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.
       

I recommend to my users to take the Airy XY resolution (conventional
optics) and divide by ~3.5. So, for Lambda = 500 nm and 1.4 NA objective
lens:

distance (Airy XY) = 0.6 * lambda / NA = 0.6 * 500 / 1.4 = 214 nm ///
divide by 3.5 for pixel size gives 61.2 nm, which I round to 60 nm (or
whatever Leica LAS AF or Zeiss ZEN rounds to).

For Z, while I would like to acquire with Zeiss or Leica's "optimized"
setting, photobleaching of most specimens leads me to usually recommend
"half overlap" (0.5 of optical slice thickness) or "layer cake" (Z-step
size equal to manufacturer recommended optical slice thickness). If I
open the pinhole, then I use layer cake (yummy).

//

Leica LAS AF owners: check out "STED/confocal deconvolution" in Process
tab. Tedious to use (I've complained about the tedious workflow - I
encourage every Leica owner to complain too). I recommend:
1. acquire as above - I use 12-bit acquisition mode [digitzer output is
12-bit on the SP5] (welcome to go for Leica recommended Z step size if
thin and/or non-bleachable specimen)
2. Generate PSF image ... FWHM = Airy distance calculated as above (214
nm for confocal, I used 80 nm for the CW-STED demo). ... I admist to not
checking the online help to see if there is a better value (of course
this whole step is part of the tedious workflow: the software should not
need to generate a stupid "dot" image at all).
2a. I use Lorentz. Why? Sounds cooler than Gauss.
3. in the STED/confocal deconv command, select the correct pair of
images (PSF and image to deconvolve), I use the default numeric value
(0.001?), and like "Signal Energy" (cooler sounding than the other
options, though unfortunately not able to make the default).
** Result image (or Z-series) is pretty quick on a cropped image. I did
a 4 channel 4kx4k Z-series (single tile) recently, did not take too long
(don't believe "95% done" on the progress meter).
    One (of many) issues: the command autoscales to the full dynamic
range brightness (i.e. 4095 for 12-bit). This makes negative controls as
well as images with bright junk look "hmmm". Best wasy to deal with it
(until Leica fixes this ... and no, it is not just "signal energy"):
Learn to contrast adjust! I hope you used modest gain (600 to 800 for
the internal SP5 PMTs, offset 0 works well for our SP5s, just above zero
intensity without any illumination).
4. Contrast adjust both the original and the STED/confocal deconv images.

Memo to Leica and other confocal manufacturers: With GPU card(s)
deconvolution (and other image processing) commands should be
instantaneous. GPU card(s) are a tiny fraction of the price of a
confocal microscope. If three people from NIST (maybe most of the heavy
lifting by one student?) can speed up 3D-SIM by 90x, the confocal and
nanoscope manufacturers ought to be able to figure out GPU programming:

    Lefman J </pubmed?term=%22Lefman%20J%22%5BAuthor%5D>, Scott K
    </pubmed?term=%22Scott%20K%22%5BAuthor%5D>, Stranick S (2011)
    </pubmed?term=%22Stranick%20S%22%5BAuthor%5D>Live, video-rate
    super-resolution microscopy using structured illumination and rapid
    GPU-based parallel processing. Microsc Microanal. <#> 2011
    Apr;17(2):191-6.

    Structured illumination fluorescence microscopy is a powerful
    super-resolution method that is capable of achieving a resolution
    below 100 nm. Each super-resolution image is computationally
    constructed from a set of differentially illuminated images.
    However, real-time application of structured illumination microscopy
    (SIM) has generally been limited due to the computational overhead
    needed to generate super-resolution images. Here, we have developed
    a real-time SIM system that incorporates graphic processing unit
    (GPU) based in-line parallel processing of raw/differentially
    illuminated images. By using GPU processing, the system has achieved
    a 90-fold increase in processing speed compared to performing
    equivalent operations on a multiprocessor computer--the total
    throughput of the system is limited by data acquisition speed, but
    not by image processing. Overall, more than 350 raw images (16-bit
    depth, 512 × 512 pixels) can be processed per second, resulting in a
    maximum frame rate of 39 super-resolution images per second. This
    ultrafast processing capability is used to provide immediate
    feedback of super-resolution images for real-time display. These
    developments are increasing the potential for sophisticated
    super-resolution imaging applications. PMID:21385522.

For another example:

    A distributed multi-GPU system for high speed electron microscopic
    tomographic reconstruction. </pubmed/21741915> Zheng SQ, Branlund E,
    Kesthelyi B, Braunfeld MB, Cheng Y, *Sedat* JW, *Agard* DA.
    Ultramicroscopy. 2011 Jul;111(8):1137-43. PMID: 21741915.




George



On 4/11/2012 8:29 AM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Renato,
>
> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.
>
> You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>
> Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.
>
> Cheers,
>
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> Having attended the first Pawley course in Vancouver I feel highly
>> embarassed to ask this, but I would really appreciate a clarification:
>>
>> When estimating the highest zoom users should apply to their sample in order
>> to accommodate for the Nyquist theorem, I estimated the optimum pixel size
>> value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
>> the value is approxiametely 90 nm.
>>
>> The doubt: if the image size is increased from 512x512 (having adjusted the
>> zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
>> (displayed by the system - Leica) the pixel size decreases 4 fold, to 22.5
>> nm. Since the resolution obviously did not change but only the image size,
>> what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>>
>> Many thanks !
>>
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara
>>      
>    

[George McNamara]

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

Hi John,

2K x 2K is a good fit to a printed page at 300 dpi ... about 7x7 inches.

See pages 25-56 of the supplemental PDF at
http://diabetes.diabetesjournals.org/content/59/4/947/suppl/DC1   for an
example.

Of course it would have been even better to post the original .LSM files
for the confocal data.

Enjoy,

George



On 4/11/2012 8:29 AM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Renato,
>
> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.
>
> You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>
> Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.
>
> Cheers,
>
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all,
>>
>> Having attended the first Pawley course in Vancouver I feel highly
>> embarassed to ask this, but I would really appreciate a clarification:
>>
>> When estimating the highest zoom users should apply to their sample in order
>> to accommodate for the Nyquist theorem, I estimated the optimum pixel size
>> value by dividing the lateral resolution (eg: 0.2 microns) by 2.3 so that
>> the value is approxiametely 90 nm.
>>
>> The doubt: if the image size is increased from 512x512 (having adjusted the
>> zoom to the pixel size of 90nm) to 2Kx2K, the resulting pixel size
>> (displayed by the system - Leica) the pixel size decreases 4 fold, to 22.5
>> nm. Since the resolution obviously did not change but only the image size,
>> what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>>
>> Many thanks !
>>
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara
>>      
>    

[Sylvie Le Guyader-2]

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

Hi everyone

"strict Nyquist is a factor of 2."

My understanding is that the Nyquist theorem is not arbitrary and that the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood well the >2 comes from this: if you want to describe a periodic signal (which is what we do when we acquire an image: we describe a sum of periodic signals), you need more than 2 points within 1 full period to collect enough information to reconstruct the periodic signal accurately. If you only give 2 points per period (e.g. only the crests and troughs), you can draw the periodic signal is several ways (e.g. double the frequency of the original signal). When we acquire an image we should thus sample more than twice the shortest period (the edges) to acquire enough information for the computer to properly reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?

Sylvie


On 11 Apr 2012, at 22:45, "David Baddeley" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> The diagonal in z will be much 'straighter' (due to the fact that the voxels are elongated in z rather than being square), making the factor much closer to 1 (probably something like 1.1) so it can safely be ignored. When talking about slightly oversampling, 2.3 is already doing this - strict Nyquist is a factor of 2. It's also worth noting that you should probably use the theoretical resolution values (ie ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and not the observed PSF width, as these reflect the bandwidth of the system. I this tend to reccommend a blanket 70x70x200nm pixel size when using a high NA objective on fixed cells. In live cells, or other delicate samples you need to exercise a little more discretion - the artefacts introduced by slight undersampling are likely to be outweighed by other considerations.
>
> My 2c,
> David
>
>
> ------------------------------
> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi John,
>>
>> Indirectly, do you suggest the same for Z sampling if we are interested in 3D measurements?  Thanks
>>
>> Monique Vasseur
>>
>> -----Message d'origine-----
>> De : Confocal Microscopy List [mailto:[hidden email]] De la part de Lemasters, John J.
>> Envoyé : 11 avril 2012 09:34
>> À : [hidden email]
>> Objet : Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.
>>
>> John
>>
>> --
>> John J. Lemasters, MD, PhD
>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury & Regeneration Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
>> DD504 Drug Discovery Building
>> 70 President Street, MSC 140
>> Charleston, SC 29425
>>
>> Office: 843-876-2360
>> Lab: 843-876-2354
>> Fax: [phone]
>> Email: [hidden email]
>> http://academicdepartments.musc.edu/ccdir
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
>> Sent: Wednesday, April 11, 2012 8:29 AM
>> To: [hidden email]
>> Subject: Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Renato,
>>
>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.
>>
>> You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>>
>> Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.
>>
>> Cheers,
>>
>>
>> John Oreopoulos
>> Research Assistant
>> Spectral Applied Research
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>>
>>
>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Dear all,
>>>
>>> Having attended the first Pawley course in Vancouver I feel highly
>>> embarassed to ask this, but I would really appreciate a clarification:
>>>
>>> When estimating the highest zoom users should apply to their sample in
>>> order to accommodate for the Nyquist theorem, I estimated the optimum
>>> pixel size value by dividing the lateral resolution (eg: 0.2 microns)
>>> by 2.3 so that the value is approxiametely 90 nm.
>>>
>>> The doubt: if the image size is increased from 512x512 (having
>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
>>> pixel size (displayed by the system - Leica) the pixel size decreases
>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change but
>>> only the image size, what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>>>
>>> Many thanks !
>>>
>>> Renato
>>>
>>> Renato A. Mortara
>>> Parasitology Division
>>> UNIFESP - Escola Paulista de Medicina
>>> Rua Botucatu, 862, 6th floor
>>> São Paulo, SP
>>> 04023-062
>>> Brazil
>>> Phone: [phone]
>>> Fax:     [phone]
>>> email: [hidden email]
>>> home page: www.ecb.epm.br/~ramortara

[Sylvie Le Guyader-2]

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

Hi everyone

"strict Nyquist is a factor of 2."

My understanding is that the Nyquist theorem is not arbitrary and that the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood well the >2 comes from this: if you want to describe a periodic signal (which is what we do when we acquire an image: we describe a sum of periodic signals), you need more than 2 points within 1 full period to collect enough information to reconstruct the periodic signal accurately. If you only give 2 points per period (e.g. only the crests and troughs), you can draw the periodic signal is several ways (e.g. double the frequency of the original signal). When we acquire an image we should thus sample more than twice the shortest period (the edges) to acquire enough information for the computer to properly reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: [phone]
LCI room: [phone]
mobile: [phone]

>
> On 11 Apr 2012, at 22:45, "David Baddeley" <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> >
> > The diagonal in z will be much 'straighter' (due to the fact that the voxels are
> elongated in z rather than being square), making the factor much closer to 1
> (probably something like 1.1) so it can safely be ignored. When talking about
> slightly oversampling, 2.3 is already doing this - strict Nyquist is a factor of 2. It's
> also worth noting that you should probably use the theoretical resolution values (ie
> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and not the
> observed PSF width, as these reflect the bandwidth of the system. I this tend to
> reccommend a blanket 70x70x200nm pixel size when using a high NA objective on
> fixed cells. In live cells, or other delicate samples you need to exercise a little more
> discretion - the artefacts introduced by slight undersampling are likely to be
> outweighed by other considerations.
> >
> > My 2c,
> > David
> >
> >
> > ------------------------------
> > On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Hi John,
> >>
> >> Indirectly, do you suggest the same for Z sampling if we are
> >> interested in 3D measurements?  Thanks
> >>
> >> Monique Vasseur
> >>
> >> -----Message d'origine-----
> >> De : Confocal Microscopy List
> [mailto:[hidden email]] De la part de Lemasters, John
> J.
> >> Envoyé : 11 avril 2012 09:34
> >> À : [hidden email]
> >> Objet : Re: Nyquist and Image size
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal
> and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal,
> pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary
> threshold, and image quality will improve somewhat with sampling greater that
> proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size
> that is one fourth of the resolving limit for the most critical work.
> >>
> >> John
> >>
> >> --
> >> John J. Lemasters, MD, PhD
> >> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
> >> Center for Cell Death, Injury & Regeneration Departments of
> >> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
> >> Biology Medical University of South Carolina
> >> DD504 Drug Discovery Building
> >> 70 President Street, MSC 140
> >> Charleston, SC 29425
> >>
> >> Office: 843-876-2360
> >> Lab: 843-876-2354
> >> Fax: [phone]
> >> Email: [hidden email]
> >> http://academicdepartments.musc.edu/ccdir
> >>
> >>
> >> -----Original Message-----
> >> From: Confocal Microscopy List
> >> [mailto:[hidden email]] On Behalf Of John
> >> Oreopoulos
> >> Sent: Wednesday, April 11, 2012 8:29 AM
> >> To: [hidden email]
> >> Subject: Re: Nyquist and Image size
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Renato,
> >>
> >> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist
> sampling rate (ie: pixel dimensions) does not change since your objective lens did
> not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm)
> means you are oversampling the image (and not gaining anything). Your image
> may look smoother but it contains no more information than the 512x512 image
> with 90x90 nm pixel sizes. Presumably the scan speed is the same between
> 512x512 and 2Kx2K.
> >>
> >> You should decrease the galvometric mirror scan zoom setting to get back to
> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively,
> you will be imaging (and properly sampling) a larger field of view then. I'm not
> familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to
> do this. On other systems, like the Olympus FV300 for example, you can set your
> image pixel dimensions (256x256, 512x512, etc.) and your scan zoom
> independently.
> >>
> >> Just out of curiosity, why image 2K x 2K when you can't easily display that on
> a standard computer screen or present it in a published paper without downsizing?
> I rarely departed from 512x512 in my laser scanning days, except when I wanted to
> see a larger field of view.
> >>
> >> Cheers,
> >>
> >>
> >> John Oreopoulos
> >> Research Assistant
> >> Spectral Applied Research
> >> Richmond Hill, Ontario
> >> Canada
> >> www.spectral.ca
> >>
> >>
> >> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>> Dear all,
> >>>
> >>> Having attended the first Pawley course in Vancouver I feel highly
> >>> embarassed to ask this, but I would really appreciate a clarification:
> >>>
> >>> When estimating the highest zoom users should apply to their sample
> >>> in order to accommodate for the Nyquist theorem, I estimated the
> >>> optimum pixel size value by dividing the lateral resolution (eg: 0.2
> >>> microns) by 2.3 so that the value is approxiametely 90 nm.
> >>>
> >>> The doubt: if the image size is increased from 512x512 (having
> >>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
> >>> pixel size (displayed by the system - Leica) the pixel size
> >>> decreases
> >>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
> >>> but only the image size, what happens to Nyquist and the optimum pixel size
> at 2Kx2K ?
> >>>
> >>> Many thanks !
> >>>
> >>> Renato
> >>>
> >>> Renato A. Mortara
> >>> Parasitology Division
> >>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
> >>> São Paulo, SP
> >>> 04023-062
> >>> Brazil
> >>> Phone: [phone]
> >>> Fax:     [phone]
> >>> email: [hidden email]
> >>> home page: www.ecb.epm.br/~ramortara

[Mark Cannell]

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

Please lets not get silly on this. The Nyquist rate is _defined_ as 2 times the bandlimit.  The Nyquist rate is defined by the sufficient condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate as David said, it does not mean Fs = 2B is sufficient!

Cheers

On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone
>
> "strict Nyquist is a factor of 2."
>
> My understanding is that the Nyquist theorem is not arbitrary and that the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood well the >2 comes from this: if you want to describe a periodic signal (which is what we do when we acquire an image: we describe a sum of periodic signals), you need more than 2 points within 1 full period to collect enough information to reconstruct the periodic signal accurately. If you only give 2 points per period (e.g. only the crests and troughs), you can draw the periodic signal is several ways (e.g. double the frequency of the original signal). When we acquire an image we should thus sample more than twice the shortest period (the edges) to acquire enough information for the computer to properly reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader
> Live Cell Imaging Unit
> Dept of Biosciences and Nutrition
> Karolinska Institutet
> Novum
> 14183 Huddinge
> Sweden
> office: [phone]
> LCI room: [phone]
> mobile: [phone]
>
>>
>> On 11 Apr 2012, at 22:45, "David Baddeley" <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>>
>>> The diagonal in z will be much 'straighter' (due to the fact that the voxels are
>> elongated in z rather than being square), making the factor much closer to 1
>> (probably something like 1.1) so it can safely be ignored. When talking about
>> slightly oversampling, 2.3 is already doing this - strict Nyquist is a factor of 2. It's
>> also worth noting that you should probably use the theoretical resolution values (ie
>> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and not the
>> observed PSF width, as these reflect the bandwidth of the system. I this tend to
>> reccommend a blanket 70x70x200nm pixel size when using a high NA objective on
>> fixed cells. In live cells, or other delicate samples you need to exercise a little more
>> discretion - the artefacts introduced by slight undersampling are likely to be
>> outweighed by other considerations.
>>>
>>> My 2c,
>>> David
>>>
>>>
>>> ------------------------------
>>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi John,
>>>>
>>>> Indirectly, do you suggest the same for Z sampling if we are
>>>> interested in 3D measurements?  Thanks
>>>>
>>>> Monique Vasseur
>>>>
>>>> -----Message d'origine-----
>>>> De : Confocal Microscopy List
>> [mailto:[hidden email]] De la part de Lemasters, John
>> J.
>>>> Envoyé : 11 avril 2012 09:34
>>>> À : [hidden email]
>>>> Objet : Re: Nyquist and Image size
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal
>> and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal,
>> pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary
>> threshold, and image quality will improve somewhat with sampling greater that
>> proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size
>> that is one fourth of the resolving limit for the most critical work.
>>>>
>>>> John
>>>>
>>>> --
>>>> John J. Lemasters, MD, PhD
>>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
>>>> Center for Cell Death, Injury & Regeneration Departments of
>>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
>>>> Biology Medical University of South Carolina
>>>> DD504 Drug Discovery Building
>>>> 70 President Street, MSC 140
>>>> Charleston, SC 29425
>>>>
>>>> Office: 843-876-2360
>>>> Lab: 843-876-2354
>>>> Fax: [phone]
>>>> Email: [hidden email]
>>>> http://academicdepartments.musc.edu/ccdir
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of John
>>>> Oreopoulos
>>>> Sent: Wednesday, April 11, 2012 8:29 AM
>>>> To: [hidden email]
>>>> Subject: Re: Nyquist and Image size
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Renato,
>>>>
>>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist
>> sampling rate (ie: pixel dimensions) does not change since your objective lens did
>> not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm)
>> means you are oversampling the image (and not gaining anything). Your image
>> may look smoother but it contains no more information than the 512x512 image
>> with 90x90 nm pixel sizes. Presumably the scan speed is the same between
>> 512x512 and 2Kx2K.
>>>>
>>>> You should decrease the galvometric mirror scan zoom setting to get back to
>> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively,
>> you will be imaging (and properly sampling) a larger field of view then. I'm not
>> familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to
>> do this. On other systems, like the Olympus FV300 for example, you can set your
>> image pixel dimensions (256x256, 512x512, etc.) and your scan zoom
>> independently.
>>>>
>>>> Just out of curiosity, why image 2K x 2K when you can't easily display that on
>> a standard computer screen or present it in a published paper without downsizing?
>> I rarely departed from 512x512 in my laser scanning days, except when I wanted to
>> see a larger field of view.
>>>>
>>>> Cheers,
>>>>
>>>>
>>>> John Oreopoulos
>>>> Research Assistant
>>>> Spectral Applied Research
>>>> Richmond Hill, Ontario
>>>> Canada
>>>> www.spectral.ca
>>>>
>>>>
>>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear all,
>>>>>
>>>>> Having attended the first Pawley course in Vancouver I feel highly
>>>>> embarassed to ask this, but I would really appreciate a clarification:
>>>>>
>>>>> When estimating the highest zoom users should apply to their sample
>>>>> in order to accommodate for the Nyquist theorem, I estimated the
>>>>> optimum pixel size value by dividing the lateral resolution (eg: 0.2
>>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>>>>>
>>>>> The doubt: if the image size is increased from 512x512 (having
>>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
>>>>> pixel size (displayed by the system - Leica) the pixel size
>>>>> decreases
>>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
>>>>> but only the image size, what happens to Nyquist and the optimum pixel size
>> at 2Kx2K ?
>>>>>
>>>>> Many thanks !
>>>>>
>>>>> Renato
>>>>>
>>>>> Renato A. Mortara
>>>>> Parasitology Division
>>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
>>>>> São Paulo, SP
>>>>> 04023-062
>>>>> Brazil
>>>>> Phone: [phone]
>>>>> Fax:     [phone]
>>>>> email: [hidden email]
>>>>> home page: www.ecb.epm.br/~ramortara

[Guy Cox-2]

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

Well, to put this in more easily understood terms, Nyquist (at 2B) defines the limit - ie the point where you cease to be able to reconstruct the wave.   So, as Mark says, you need to get beyond this to actually be able to get information.  The often-quoted 2.3B more or less corresponds to the Rayleigh resolution criterion,  ie the point at which you can reconstruct the wave at usable contrast.  However, the other problem we face is that we do NOT reconstruct the sine wave, we just look at a map of little squares.  This is stupid.  

Required reading should be:

A Pixel Is Not A Little Square,
A Pixel Is Not A Little Square,
A Pixel Is Not A Little Square!
(And a Voxel is Not a Little Cube)
Microsoft Technical Memo 6
Alvy Ray Smith
July 17, 1995

(Yes, that really is the title)

It's on the Microsoft web site, or I can mail a copy to anyone who is interested.  

                                                                                              Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mark Cannell
Sent: Friday, 13 April 2012 5:53 PM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Please lets not get silly on this. The Nyquist rate is _defined_ as 2 times the bandlimit.  The Nyquist rate is defined by the sufficient condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate as David said, it does not mean Fs = 2B is sufficient!

Cheers

On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi everyone
>
> "strict Nyquist is a factor of 2."
>
> My understanding is that the Nyquist theorem is not arbitrary and that the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood well the >2 comes from this: if you want to describe a periodic signal (which is what we do when we acquire an image: we describe a sum of periodic signals), you need more than 2 points within 1 full period to collect enough information to reconstruct the periodic signal accurately. If you only give 2 points per period (e.g. only the crests and troughs), you can draw the periodic signal is several ways (e.g. double the frequency of the original signal). When we acquire an image we should thus sample more than twice the shortest period (the edges) to acquire enough information for the computer to properly reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
> Sylvie Le Guyader
> Live Cell Imaging Unit
> Dept of Biosciences and Nutrition
> Karolinska Institutet
> Novum
> 14183 Huddinge
> Sweden
> office: [phone]
> LCI room: [phone]
> mobile: [phone]
>
>>
>> On 11 Apr 2012, at 22:45, "David Baddeley"
>> <[hidden email]>
>> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>>
>>> The diagonal in z will be much 'straighter' (due to the fact that
>>> the voxels are
>> elongated in z rather than being square), making the factor much
>> closer to 1 (probably something like 1.1) so it can safely be
>> ignored. When talking about slightly oversampling, 2.3 is already
>> doing this - strict Nyquist is a factor of 2. It's also worth noting
>> that you should probably use the theoretical resolution values (ie
>> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and
>> not the observed PSF width, as these reflect the bandwidth of the
>> system. I this tend to reccommend a blanket 70x70x200nm pixel size
>> when using a high NA objective on fixed cells. In live cells, or
>> other delicate samples you need to exercise a little more discretion
>> - the artefacts introduced by slight undersampling are likely to be outweighed by other considerations.
>>>
>>> My 2c,
>>> David
>>>
>>>
>>> ------------------------------
>>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi John,
>>>>
>>>> Indirectly, do you suggest the same for Z sampling if we are
>>>> interested in 3D measurements?  Thanks
>>>>
>>>> Monique Vasseur
>>>>
>>>> -----Message d'origine-----
>>>> De : Confocal Microscopy List
>> [mailto:[hidden email]] De la part de Lemasters,
>> John J.
>>>> Envoyé : 11 avril 2012 09:34
>>>> À : [hidden email] Objet : Re: Nyquist and Image
>>>> size
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Please remember that pixel spacing on the diagonal is 1.4 that in
>>>> the horizontal
>> and vertical directions. Accordingly to meet the Nyquist criterion
>> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>> Nyquist criterion is an arbitrary threshold, and image quality will
>> improve somewhat with sampling greater that proposed by Nyquist.
>> Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.
>>>>
>>>> John
>>>>
>>>> --
>>>> John J. Lemasters, MD, PhD
>>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
>>>> Center for Cell Death, Injury & Regeneration Departments of
>>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
>>>> Biology Medical University of South Carolina
>>>> DD504 Drug Discovery Building
>>>> 70 President Street, MSC 140
>>>> Charleston, SC 29425
>>>>
>>>> Office: 843-876-2360
>>>> Lab: 843-876-2354
>>>> Fax: [phone]
>>>> Email: [hidden email]
>>>> http://academicdepartments.musc.edu/ccdir
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List
>>>> [mailto:[hidden email]] On Behalf Of John
>>>> Oreopoulos
>>>> Sent: Wednesday, April 11, 2012 8:29 AM
>>>> To: [hidden email]
>>>> Subject: Re: Nyquist and Image size
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Renato,
>>>>
>>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>>>> Nyquist
>> sampling rate (ie: pixel dimensions) does not change since your
>> objective lens did not change. The quoted pixel size at 2Kx2K you
>> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>> (and not gaining anything). Your image may look smoother but it
>> contains no more information than the 512x512 image with 90x90 nm
>> pixel sizes. Presumably the scan speed is the same between
>> 512x512 and 2Kx2K.
>>>>
>>>> You should decrease the galvometric mirror scan zoom setting to get
>>>> back to
>> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>> Effectively, you will be imaging (and properly sampling) a larger
>> field of view then. I'm not familiar with the Leica laser scanning
>> confocals so I'm not sure if it will allow you to do this. On other
>> systems, like the Olympus FV300 for example, you can set your image
>> pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>>>>
>>>> Just out of curiosity, why image 2K x 2K when you can't easily
>>>> display that on
>> a standard computer screen or present it in a published paper without downsizing?
>> I rarely departed from 512x512 in my laser scanning days, except when
>> I wanted to see a larger field of view.
>>>>
>>>> Cheers,
>>>>
>>>>
>>>> John Oreopoulos
>>>> Research Assistant
>>>> Spectral Applied Research
>>>> Richmond Hill, Ontario
>>>> Canada
>>>> www.spectral.ca
>>>>
>>>>
>>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> Dear all,
>>>>>
>>>>> Having attended the first Pawley course in Vancouver I feel highly
>>>>> embarassed to ask this, but I would really appreciate a clarification:
>>>>>
>>>>> When estimating the highest zoom users should apply to their
>>>>> sample in order to accommodate for the Nyquist theorem, I
>>>>> estimated the optimum pixel size value by dividing the lateral
>>>>> resolution (eg: 0.2
>>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>>>>>
>>>>> The doubt: if the image size is increased from 512x512 (having
>>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>>>>> resulting pixel size (displayed by the system - Leica) the pixel
>>>>> size decreases
>>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
>>>>> but only the image size, what happens to Nyquist and the optimum
>>>>> pixel size
>> at 2Kx2K ?
>>>>>
>>>>> Many thanks !
>>>>>
>>>>> Renato
>>>>>
>>>>> Renato A. Mortara
>>>>> Parasitology Division
>>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
>>>>> São Paulo, SP
>>>>> 04023-062
>>>>> Brazil
>>>>> Phone: [phone]
>>>>> Fax:     [phone]
>>>>> email: [hidden email]
>>>>> home page: www.ecb.epm.br/~ramortara

[JOEL B. SHEFFIELD]

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

Excellent points.  That paper is a joy to read.
Joel


On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Well, to put this in more easily understood terms, Nyquist (at 2B) defines
> the limit - ie the point where you cease to be able to reconstruct the
> wave.   So, as Mark says, you need to get beyond this to actually be able
> to get information.  The often-quoted 2.3B more or less corresponds to the
> Rayleigh resolution criterion,  ie the point at which you can reconstruct
> the wave at usable contrast.  However, the other problem we face is that we
> do NOT reconstruct the sine wave, we just look at a map of little squares.
>  This is stupid.
>
> Required reading should be:
>
> A Pixel Is Not A Little Square,
> A Pixel Is Not A Little Square,
> A Pixel Is Not A Little Square!
> (And a Voxel is Not a Little Cube)
> Microsoft Technical Memo 6
> Alvy Ray Smith
> July 17, 1995
>
> (Yes, that really is the title)
>
> It's on the Microsoft web site, or I can mail a copy to anyone who is
> interested.
>
>
>                    Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: Friday, 13 April 2012 5:53 PM
> To: [hidden email]
> Subject: Re: Nyquist and Image size
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
> times the bandlimit.  The Nyquist rate is defined by the sufficient
> condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate
> as David said, it does not mean Fs = 2B is sufficient!
>
> Cheers
>
> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi everyone
> >
> > "strict Nyquist is a factor of 2."
> >
> > My understanding is that the Nyquist theorem is not arbitrary and that
> the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood
> well the >2 comes from this: if you want to describe a periodic signal
> (which is what we do when we acquire an image: we describe a sum of
> periodic signals), you need more than 2 points within 1 full period to
> collect enough information to reconstruct the periodic signal accurately.
> If you only give 2 points per period (e.g. only the crests and troughs),
> you can draw the periodic signal is several ways (e.g. double the frequency
> of the original signal). When we acquire an image we should thus sample
> more than twice the shortest period (the edges) to acquire enough
> information for the computer to properly reconstruct the image. This is why
> the Nyquist criterion is 'more than 2'. Am I right?
> >
> > Sylvie
> >
> > @@@@@@@@@@@@@@@@@@@@@@@@
> > Sylvie Le Guyader
> > Live Cell Imaging Unit
> > Dept of Biosciences and Nutrition
> > Karolinska Institutet
> > Novum
> > 14183 Huddinge
> > Sweden
> > office: [phone]
> > LCI room: [phone]
> > mobile: [phone]
> >
> >>
> >> On 11 Apr 2012, at 22:45, "David Baddeley"
> >> <[hidden email]>
> >> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> *****
> >>>
> >>>
> >>> The diagonal in z will be much 'straighter' (due to the fact that
> >>> the voxels are
> >> elongated in z rather than being square), making the factor much
> >> closer to 1 (probably something like 1.1) so it can safely be
> >> ignored. When talking about slightly oversampling, 2.3 is already
> >> doing this - strict Nyquist is a factor of 2. It's also worth noting
> >> that you should probably use the theoretical resolution values (ie
> >> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and
> >> not the observed PSF width, as these reflect the bandwidth of the
> >> system. I this tend to reccommend a blanket 70x70x200nm pixel size
> >> when using a high NA objective on fixed cells. In live cells, or
> >> other delicate samples you need to exercise a little more discretion
> >> - the artefacts introduced by slight undersampling are likely to be
> outweighed by other considerations.
> >>>
> >>> My 2c,
> >>> David
> >>>
> >>>
> >>> ------------------------------
> >>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
> >>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>>> *****
> >>>>
> >>>> Hi John,
> >>>>
> >>>> Indirectly, do you suggest the same for Z sampling if we are
> >>>> interested in 3D measurements?  Thanks
> >>>>
> >>>> Monique Vasseur
> >>>>
> >>>> -----Message d'origine-----
> >>>> De : Confocal Microscopy List
> >> [mailto:[hidden email]] De la part de Lemasters,
> >> John J.
> >>>> Envoyé : 11 avril 2012 09:34
> >>>> À : [hidden email] Objet : Re: Nyquist and Image
> >>>> size
> >>>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>>> *****
> >>>>
> >>>> Please remember that pixel spacing on the diagonal is 1.4 that in
> >>>> the horizontal
> >> and vertical directions. Accordingly to meet the Nyquist criterion
> >> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
> >> Nyquist criterion is an arbitrary threshold, and image quality will
> >> improve somewhat with sampling greater that proposed by Nyquist.
> >> Considering diagonal sampling, I suggest using a pixel size that is one
> fourth of the resolving limit for the most critical work.
> >>>>
> >>>> John
> >>>>
> >>>> --
> >>>> John J. Lemasters, MD, PhD
> >>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
> >>>> Center for Cell Death, Injury & Regeneration Departments of
> >>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
> >>>> Biology Medical University of South Carolina
> >>>> DD504 Drug Discovery Building
> >>>> 70 President Street, MSC 140
> >>>> Charleston, SC 29425
> >>>>
> >>>> Office: 843-876-2360
> >>>> Lab: 843-876-2354
> >>>> Fax: [phone]
> >>>> Email: [hidden email]
> >>>> http://academicdepartments.musc.edu/ccdir
> >>>>
> >>>>
> >>>> -----Original Message-----
> >>>> From: Confocal Microscopy List
> >>>> [mailto:[hidden email]] On Behalf Of John
> >>>> Oreopoulos
> >>>> Sent: Wednesday, April 11, 2012 8:29 AM
> >>>> To: [hidden email]
> >>>> Subject: Re: Nyquist and Image size
> >>>>
> >>>> *****
> >>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>>> *****
> >>>>
> >>>> Renato,
> >>>>
> >>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
> >>>> Nyquist
> >> sampling rate (ie: pixel dimensions) does not change since your
> >> objective lens did not change. The quoted pixel size at 2Kx2K you
> >> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
> >> (and not gaining anything). Your image may look smoother but it
> >> contains no more information than the 512x512 image with 90x90 nm
> >> pixel sizes. Presumably the scan speed is the same between
> >> 512x512 and 2Kx2K.
> >>>>
> >>>> You should decrease the galvometric mirror scan zoom setting to get
> >>>> back to
> >> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
> >> Effectively, you will be imaging (and properly sampling) a larger
> >> field of view then. I'm not familiar with the Leica laser scanning
> >> confocals so I'm not sure if it will allow you to do this. On other
> >> systems, like the Olympus FV300 for example, you can set your image
> >> pixel dimensions (256x256, 512x512, etc.) and your scan zoom
> independently.
> >>>>
> >>>> Just out of curiosity, why image 2K x 2K when you can't easily
> >>>> display that on
> >> a standard computer screen or present it in a published paper without
> downsizing?
> >> I rarely departed from 512x512 in my laser scanning days, except when
> >> I wanted to see a larger field of view.
> >>>>
> >>>> Cheers,
> >>>>
> >>>>
> >>>> John Oreopoulos
> >>>> Research Assistant
> >>>> Spectral Applied Research
> >>>> Richmond Hill, Ontario
> >>>> Canada
> >>>> www.spectral.ca
> >>>>
> >>>>
> >>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
> >>>>
> >>>>> *****
> >>>>> To join, leave or search the confocal microscopy listserv, go to:
> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>>>> *****
> >>>>>
> >>>>> Dear all,
> >>>>>
> >>>>> Having attended the first Pawley course in Vancouver I feel highly
> >>>>> embarassed to ask this, but I would really appreciate a
> clarification:
> >>>>>
> >>>>> When estimating the highest zoom users should apply to their
> >>>>> sample in order to accommodate for the Nyquist theorem, I
> >>>>> estimated the optimum pixel size value by dividing the lateral
> >>>>> resolution (eg: 0.2
> >>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
> >>>>>
> >>>>> The doubt: if the image size is increased from 512x512 (having
> >>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
> >>>>> resulting pixel size (displayed by the system - Leica) the pixel
> >>>>> size decreases
> >>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
> >>>>> but only the image size, what happens to Nyquist and the optimum
> >>>>> pixel size
> >> at 2Kx2K ?
> >>>>>
> >>>>> Many thanks !
> >>>>>
> >>>>> Renato
> >>>>>
> >>>>> Renato A. Mortara
> >>>>> Parasitology Division
> >>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
> >>>>> São Paulo, SP
> >>>>> 04023-062
> >>>>> Brazil
> >>>>> Phone: [phone]
> >>>>> Fax:     [phone]
> >>>>> email: [hidden email]
> >>>>> home page: www.ecb.epm.br/~ramortara<http://www.ecb.epm.br/%7Eramortara>
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[Renato A. Mortara]

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

Hi Guy, it would be great if you could email the article to me:

Thanks again for all the inputs !

Renato


Renato A. Mortara
Disciplina de Parasitologia
UNIFESP Escola Paulista de Medicina
R. Botucatu, 862 6o andar
04023-062
São Paulo SP
Brasil
[hidden email]


Citando "Joel B. Sheffield" <[hidden email]>:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Excellent points.  That paper is a joy to read.
> Joel
>
>
> On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, to put this in more easily understood terms, Nyquist (at 2B) defines
>> the limit - ie the point where you cease to be able to reconstruct the
>> wave.   So, as Mark says, you need to get beyond this to actually be able
>> to get information.  The often-quoted 2.3B more or less corresponds to the
>> Rayleigh resolution criterion,  ie the point at which you can reconstruct
>> the wave at usable contrast.  However, the other problem we face is that we
>> do NOT reconstruct the sine wave, we just look at a map of little squares.
>>  This is stupid.
>>
>> Required reading should be:
>>
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square!
>> (And a Voxel is Not a Little Cube)
>> Microsoft Technical Memo 6
>> Alvy Ray Smith
>> July 17, 1995
>>
>> (Yes, that really is the title)
>>
>> It's on the Microsoft web site, or I can mail a copy to anyone who is
>> interested.
>>
>>
>>                    Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 April 2012 5:53 PM
>> To: [hidden email]
>> Subject: Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
>> times the bandlimit.  The Nyquist rate is defined by the sufficient
>> condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate
>> as David said, it does not mean Fs = 2B is sufficient!
>>
>> Cheers
>>
>> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Hi everyone
>> >
>> > "strict Nyquist is a factor of 2."
>> >
>> > My understanding is that the Nyquist theorem is not arbitrary and that
>> the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood
>> well the >2 comes from this: if you want to describe a periodic signal
>> (which is what we do when we acquire an image: we describe a sum of
>> periodic signals), you need more than 2 points within 1 full period to
>> collect enough information to reconstruct the periodic signal accurately.
>> If you only give 2 points per period (e.g. only the crests and troughs),
>> you can draw the periodic signal is several ways (e.g. double the frequency
>> of the original signal). When we acquire an image we should thus sample
>> more than twice the shortest period (the edges) to acquire enough
>> information for the computer to properly reconstruct the image. This is why
>> the Nyquist criterion is 'more than 2'. Am I right?
>> >
>> > Sylvie
>> >
>> > @@@@@@@@@@@@@@@@@@@@@@@@
>> > Sylvie Le Guyader
>> > Live Cell Imaging Unit
>> > Dept of Biosciences and Nutrition
>> > Karolinska Institutet
>> > Novum
>> > 14183 Huddinge
>> > Sweden
>> > office: [phone]
>> > LCI room: [phone]
>> > mobile: [phone]
>> >
>> >>
>> >> On 11 Apr 2012, at 22:45, "David Baddeley"
>> >> <[hidden email]>
>> >> wrote:
>> >>
>> >>> *****
>> >>> To join, leave or search the confocal microscopy listserv, go to:
>> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>> *****
>> >>>
>> >>>
>> >>> The diagonal in z will be much 'straighter' (due to the fact that
>> >>> the voxels are
>> >> elongated in z rather than being square), making the factor much
>> >> closer to 1 (probably something like 1.1) so it can safely be
>> >> ignored. When talking about slightly oversampling, 2.3 is already
>> >> doing this - strict Nyquist is a factor of 2. It's also worth noting
>> >> that you should probably use the theoretical resolution values (ie
>> >> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and
>> >> not the observed PSF width, as these reflect the bandwidth of the
>> >> system. I this tend to reccommend a blanket 70x70x200nm pixel size
>> >> when using a high NA objective on fixed cells. In live cells, or
>> >> other delicate samples you need to exercise a little more discretion
>> >> - the artefacts introduced by slight undersampling are likely to be
>> outweighed by other considerations.
>> >>>
>> >>> My 2c,
>> >>> David
>> >>>
>> >>>
>> >>> ------------------------------
>> >>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>> >>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Hi John,
>> >>>>
>> >>>> Indirectly, do you suggest the same for Z sampling if we are
>> >>>> interested in 3D measurements?  Thanks
>> >>>>
>> >>>> Monique Vasseur
>> >>>>
>> >>>> -----Message d'origine-----
>> >>>> De : Confocal Microscopy List
>> >> [mailto:[hidden email]] De la part de Lemasters,
>> >> John J.
>> >>>> Envoyé : 11 avril 2012 09:34
>> >>>> À : [hidden email] Objet : Re: Nyquist and Image
>> >>>> size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Please remember that pixel spacing on the diagonal is 1.4 that in
>> >>>> the horizontal
>> >> and vertical directions. Accordingly to meet the Nyquist criterion
>> >> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>> >> Nyquist criterion is an arbitrary threshold, and image quality will
>> >> improve somewhat with sampling greater that proposed by Nyquist.
>> >> Considering diagonal sampling, I suggest using a pixel size that is one
>> fourth of the resolving limit for the most critical work.
>> >>>>
>> >>>> John
>> >>>>
>> >>>> --
>> >>>> John J. Lemasters, MD, PhD
>> >>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
>> >>>> Center for Cell Death, Injury & Regeneration Departments of
>> >>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
>> >>>> Biology Medical University of South Carolina
>> >>>> DD504 Drug Discovery Building
>> >>>> 70 President Street, MSC 140
>> >>>> Charleston, SC 29425
>> >>>>
>> >>>> Office: 843-876-2360
>> >>>> Lab: 843-876-2354
>> >>>> Fax: [phone]
>> >>>> Email: [hidden email]
>> >>>> http://academicdepartments.musc.edu/ccdir
>> >>>>
>> >>>>
>> >>>> -----Original Message-----
>> >>>> From: Confocal Microscopy List
>> >>>> [mailto:[hidden email]] On Behalf Of John
>> >>>> Oreopoulos
>> >>>> Sent: Wednesday, April 11, 2012 8:29 AM
>> >>>> To: [hidden email]
>> >>>> Subject: Re: Nyquist and Image size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Renato,
>> >>>>
>> >>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>> >>>> Nyquist
>> >> sampling rate (ie: pixel dimensions) does not change since your
>> >> objective lens did not change. The quoted pixel size at 2Kx2K you
>> >> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>> >> (and not gaining anything). Your image may look smoother but it
>> >> contains no more information than the 512x512 image with 90x90 nm
>> >> pixel sizes. Presumably the scan speed is the same between
>> >> 512x512 and 2Kx2K.
>> >>>>
>> >>>> You should decrease the galvometric mirror scan zoom setting to get
>> >>>> back to
>> >> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>> >> Effectively, you will be imaging (and properly sampling) a larger
>> >> field of view then. I'm not familiar with the Leica laser scanning
>> >> confocals so I'm not sure if it will allow you to do this. On other
>> >> systems, like the Olympus FV300 for example, you can set your image
>> >> pixel dimensions (256x256, 512x512, etc.) and your scan zoom
>> independently.
>> >>>>
>> >>>> Just out of curiosity, why image 2K x 2K when you can't easily
>> >>>> display that on
>> >> a standard computer screen or present it in a published paper without
>> downsizing?
>> >> I rarely departed from 512x512 in my laser scanning days, except when
>> >> I wanted to see a larger field of view.
>> >>>>
>> >>>> Cheers,
>> >>>>
>> >>>>
>> >>>> John Oreopoulos
>> >>>> Research Assistant
>> >>>> Spectral Applied Research
>> >>>> Richmond Hill, Ontario
>> >>>> Canada
>> >>>> www.spectral.ca
>> >>>>
>> >>>>
>> >>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>> >>>>
>> >>>>> *****
>> >>>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>>> *****
>> >>>>>
>> >>>>> Dear all,
>> >>>>>
>> >>>>> Having attended the first Pawley course in Vancouver I feel highly
>> >>>>> embarassed to ask this, but I would really appreciate a
>> clarification:
>> >>>>>
>> >>>>> When estimating the highest zoom users should apply to their
>> >>>>> sample in order to accommodate for the Nyquist theorem, I
>> >>>>> estimated the optimum pixel size value by dividing the lateral
>> >>>>> resolution (eg: 0.2
>> >>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>> >>>>>
>> >>>>> The doubt: if the image size is increased from 512x512 (having
>> >>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>> >>>>> resulting pixel size (displayed by the system - Leica) the pixel
>> >>>>> size decreases
>> >>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
>> >>>>> but only the image size, what happens to Nyquist and the optimum
>> >>>>> pixel size
>> >> at 2Kx2K ?
>> >>>>>
>> >>>>> Many thanks !
>> >>>>>
>> >>>>> Renato
>> >>>>>
>> >>>>> Renato A. Mortara
>> >>>>> Parasitology Division
>> >>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
>> >>>>> São Paulo, SP
>> >>>>> 04023-062
>> >>>>> Brazil
>> >>>>> Phone: [phone]
>> >>>>> Fax:     [phone]
>> >>>>> email: [hidden email]
>> >>>>> home page:  
>> www.ecb.epm.br/~ramortara<http://www.ecb.epm.br/%7Eramortara>
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>
>

[Armstrong, Brian]

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

Thanks for the paper Guy, it is amusing and informative. I always wondered how to get that .3 of a pixel!

Back to the original question; perhaps you could explain to the users not to zoom more than 1000x NA. This is a more straight forward conversation than explaining the Nyquist/Shannon theorem to nascent Confocal users. I often see users applying too much zoom and collecting empty magnification.
 
Numbers: using a 20x/0.8NA lens and lambda of 500nm AU=1, Nyquist is ~ 165nm. If you use 4x optical zoom, you have 200x X 4 = 800x (1000 X NA of 0.8 = 800x) which at 1024x1024 gives you a pixel size of X,Y = 110nm which is comfortably under Nyquist sampling.

There is not usually a reason to zoom more than 3x on an CLSM.

Cheers,

Brian D Armstrong PhD
Assistant Research Professor
Director, Light Microscopy Core
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


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato A. Mortara
Sent: Friday, April 13, 2012 9:13 AM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Hi Guy, it would be great if you could email the article to me:

Thanks again for all the inputs !

Renato


Renato A. Mortara
Disciplina de Parasitologia
UNIFESP Escola Paulista de Medicina
R. Botucatu, 862 6o andar
04023-062
São Paulo SP
Brasil
[hidden email]


Citando "Joel B. Sheffield" <[hidden email]>:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Excellent points.  That paper is a joy to read.
> Joel
>
>
> On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, to put this in more easily understood terms, Nyquist (at 2B) defines
>> the limit - ie the point where you cease to be able to reconstruct the
>> wave.   So, as Mark says, you need to get beyond this to actually be able
>> to get information.  The often-quoted 2.3B more or less corresponds to the
>> Rayleigh resolution criterion,  ie the point at which you can reconstruct
>> the wave at usable contrast.  However, the other problem we face is that we
>> do NOT reconstruct the sine wave, we just look at a map of little squares.
>>  This is stupid.
>>
>> Required reading should be:
>>
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square!
>> (And a Voxel is Not a Little Cube)
>> Microsoft Technical Memo 6
>> Alvy Ray Smith
>> July 17, 1995
>>
>> (Yes, that really is the title)
>>
>> It's on the Microsoft web site, or I can mail a copy to anyone who is
>> interested.
>>
>>
>>                    Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 April 2012 5:53 PM
>> To: [hidden email]
>> Subject: Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
>> times the bandlimit.  The Nyquist rate is defined by the sufficient
>> condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate
>> as David said, it does not mean Fs = 2B is sufficient!
>>
>> Cheers
>>
>> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Hi everyone
>> >
>> > "strict Nyquist is a factor of 2."
>> >
>> > My understanding is that the Nyquist theorem is not arbitrary and that
>> the factor is actually >2. So 2.1 would do as well as 2.3.  If i understood
>> well the >2 comes from this: if you want to describe a periodic signal
>> (which is what we do when we acquire an image: we describe a sum of
>> periodic signals), you need more than 2 points within 1 full period to
>> collect enough information to reconstruct the periodic signal accurately.
>> If you only give 2 points per period (e.g. only the crests and troughs),
>> you can draw the periodic signal is several ways (e.g. double the frequency
>> of the original signal). When we acquire an image we should thus sample
>> more than twice the shortest period (the edges) to acquire enough
>> information for the computer to properly reconstruct the image. This is why
>> the Nyquist criterion is 'more than 2'. Am I right?
>> >
>> > Sylvie
>> >
>> > @@@@@@@@@@@@@@@@@@@@@@@@
>> > Sylvie Le Guyader
>> > Live Cell Imaging Unit
>> > Dept of Biosciences and Nutrition
>> > Karolinska Institutet
>> > Novum
>> > 14183 Huddinge
>> > Sweden
>> > office: [phone]
>> > LCI room: [phone]
>> > mobile: [phone]
>> >
>> >>
>> >> On 11 Apr 2012, at 22:45, "David Baddeley"
>> >> <[hidden email]>
>> >> wrote:
>> >>
>> >>> *****
>> >>> To join, leave or search the confocal microscopy listserv, go to:
>> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>> *****
>> >>>
>> >>>
>> >>> The diagonal in z will be much 'straighter' (due to the fact that
>> >>> the voxels are
>> >> elongated in z rather than being square), making the factor much
>> >> closer to 1 (probably something like 1.1) so it can safely be
>> >> ignored. When talking about slightly oversampling, 2.3 is already
>> >> doing this - strict Nyquist is a factor of 2. It's also worth noting
>> >> that you should probably use the theoretical resolution values (ie
>> >> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and
>> >> not the observed PSF width, as these reflect the bandwidth of the
>> >> system. I this tend to reccommend a blanket 70x70x200nm pixel size
>> >> when using a high NA objective on fixed cells. In live cells, or
>> >> other delicate samples you need to exercise a little more discretion
>> >> - the artefacts introduced by slight undersampling are likely to be
>> outweighed by other considerations.
>> >>>
>> >>> My 2c,
>> >>> David
>> >>>
>> >>>
>> >>> ------------------------------
>> >>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>> >>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Hi John,
>> >>>>
>> >>>> Indirectly, do you suggest the same for Z sampling if we are
>> >>>> interested in 3D measurements?  Thanks
>> >>>>
>> >>>> Monique Vasseur
>> >>>>
>> >>>> -----Message d'origine-----
>> >>>> De : Confocal Microscopy List
>> >> [mailto:[hidden email]] De la part de Lemasters,
>> >> John J.
>> >>>> Envoyé : 11 avril 2012 09:34
>> >>>> À : [hidden email] Objet : Re: Nyquist and Image
>> >>>> size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Please remember that pixel spacing on the diagonal is 1.4 that in
>> >>>> the horizontal
>> >> and vertical directions. Accordingly to meet the Nyquist criterion
>> >> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>> >> Nyquist criterion is an arbitrary threshold, and image quality will
>> >> improve somewhat with sampling greater that proposed by Nyquist.
>> >> Considering diagonal sampling, I suggest using a pixel size that is one
>> fourth of the resolving limit for the most critical work.
>> >>>>
>> >>>> John
>> >>>>
>> >>>> --
>> >>>> John J. Lemasters, MD, PhD
>> >>>> Professor and GlaxoSmithKline Distinguished Endowed Chair Director,
>> >>>> Center for Cell Death, Injury & Regeneration Departments of
>> >>>> Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular
>> >>>> Biology Medical University of South Carolina
>> >>>> DD504 Drug Discovery Building
>> >>>> 70 President Street, MSC 140
>> >>>> Charleston, SC 29425
>> >>>>
>> >>>> Office: 843-876-2360
>> >>>> Lab: 843-876-2354
>> >>>> Fax: [phone]
>> >>>> Email: [hidden email]
>> >>>> http://academicdepartments.musc.edu/ccdir
>> >>>>
>> >>>>
>> >>>> -----Original Message-----
>> >>>> From: Confocal Microscopy List
>> >>>> [mailto:[hidden email]] On Behalf Of John
>> >>>> Oreopoulos
>> >>>> Sent: Wednesday, April 11, 2012 8:29 AM
>> >>>> To: [hidden email]
>> >>>> Subject: Re: Nyquist and Image size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Renato,
>> >>>>
>> >>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>> >>>> Nyquist
>> >> sampling rate (ie: pixel dimensions) does not change since your
>> >> objective lens did not change. The quoted pixel size at 2Kx2K you
>> >> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>> >> (and not gaining anything). Your image may look smoother but it
>> >> contains no more information than the 512x512 image with 90x90 nm
>> >> pixel sizes. Presumably the scan speed is the same between
>> >> 512x512 and 2Kx2K.
>> >>>>
>> >>>> You should decrease the galvometric mirror scan zoom setting to get
>> >>>> back to
>> >> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>> >> Effectively, you will be imaging (and properly sampling) a larger
>> >> field of view then. I'm not familiar with the Leica laser scanning
>> >> confocals so I'm not sure if it will allow you to do this. On other
>> >> systems, like the Olympus FV300 for example, you can set your image
>> >> pixel dimensions (256x256, 512x512, etc.) and your scan zoom
>> independently.
>> >>>>
>> >>>> Just out of curiosity, why image 2K x 2K when you can't easily
>> >>>> display that on
>> >> a standard computer screen or present it in a published paper without
>> downsizing?
>> >> I rarely departed from 512x512 in my laser scanning days, except when
>> >> I wanted to see a larger field of view.
>> >>>>
>> >>>> Cheers,
>> >>>>
>> >>>>
>> >>>> John Oreopoulos
>> >>>> Research Assistant
>> >>>> Spectral Applied Research
>> >>>> Richmond Hill, Ontario
>> >>>> Canada
>> >>>> www.spectral.ca
>> >>>>
>> >>>>
>> >>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>> >>>>
>> >>>>> *****
>> >>>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>>> *****
>> >>>>>
>> >>>>> Dear all,
>> >>>>>
>> >>>>> Having attended the first Pawley course in Vancouver I feel highly
>> >>>>> embarassed to ask this, but I would really appreciate a
>> clarification:
>> >>>>>
>> >>>>> When estimating the highest zoom users should apply to their
>> >>>>> sample in order to accommodate for the Nyquist theorem, I
>> >>>>> estimated the optimum pixel size value by dividing the lateral
>> >>>>> resolution (eg: 0.2
>> >>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>> >>>>>
>> >>>>> The doubt: if the image size is increased from 512x512 (having
>> >>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>> >>>>> resulting pixel size (displayed by the system - Leica) the pixel
>> >>>>> size decreases
>> >>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not change
>> >>>>> but only the image size, what happens to Nyquist and the optimum
>> >>>>> pixel size
>> >> at 2Kx2K ?
>> >>>>>
>> >>>>> Many thanks !
>> >>>>>
>> >>>>> Renato
>> >>>>>
>> >>>>> Renato A. Mortara
>> >>>>> Parasitology Division
>> >>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th floor
>> >>>>> São Paulo, SP
>> >>>>> 04023-062
>> >>>>> Brazil
>> >>>>> Phone: [phone]
>> >>>>> Fax:     [phone]
>> >>>>> email: [hidden email]
>> >>>>> home page:  
>> www.ecb.epm.br/~ramortara<http://www.ecb.epm.br/%7Eramortara>
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>
>

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[Guy Cox-2]

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

http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato A. Mortara
Sent: Saturday, 14 April 2012 2:13 AM
To: [hidden email]
Subject: Re: Nyquist and Image size

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

Hi Guy, it would be great if you could email the article to me:

Thanks again for all the inputs !

Renato


Renato A. Mortara
Disciplina de Parasitologia
UNIFESP Escola Paulista de Medicina
R. Botucatu, 862 6o andar
04023-062
São Paulo SP
Brasil
[hidden email]


Citando "Joel B. Sheffield" <[hidden email]>:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Excellent points.  That paper is a joy to read.
> Joel
>
>
> On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Well, to put this in more easily understood terms, Nyquist (at 2B)
>> defines the limit - ie the point where you cease to be able to reconstruct the
>> wave.   So, as Mark says, you need to get beyond this to actually be able
>> to get information.  The often-quoted 2.3B more or less corresponds
>> to the Rayleigh resolution criterion,  ie the point at which you can
>> reconstruct the wave at usable contrast.  However, the other problem
>> we face is that we do NOT reconstruct the sine wave, we just look at a map of little squares.
>>  This is stupid.
>>
>> Required reading should be:
>>
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square,
>> A Pixel Is Not A Little Square!
>> (And a Voxel is Not a Little Cube)
>> Microsoft Technical Memo 6
>> Alvy Ray Smith
>> July 17, 1995
>>
>> (Yes, that really is the title)
>>
>> It's on the Microsoft web site, or I can mail a copy to anyone who is
>> interested.
>>
>>
>>                    Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List
>> [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 April 2012 5:53 PM
>> To: [hidden email]
>> Subject: Re: Nyquist and Image size
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
>> times the bandlimit.  The Nyquist rate is defined by the sufficient
>> condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate
>> as David said, it does not mean Fs = 2B is sufficient!
>>
>> Cheers
>>
>> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Hi everyone
>> >
>> > "strict Nyquist is a factor of 2."
>> >
>> > My understanding is that the Nyquist theorem is not arbitrary and
>> > that
>> the factor is actually >2. So 2.1 would do as well as 2.3.  If i
>> understood well the >2 comes from this: if you want to describe a
>> periodic signal (which is what we do when we acquire an image: we
>> describe a sum of periodic signals), you need more than 2 points
>> within 1 full period to collect enough information to reconstruct the periodic signal accurately.
>> If you only give 2 points per period (e.g. only the crests and
>> troughs), you can draw the periodic signal is several ways (e.g.
>> double the frequency of the original signal). When we acquire an
>> image we should thus sample more than twice the shortest period (the
>> edges) to acquire enough information for the computer to properly
>> reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?
>> >
>> > Sylvie
>> >
>> > @@@@@@@@@@@@@@@@@@@@@@@@
>> > Sylvie Le Guyader
>> > Live Cell Imaging Unit
>> > Dept of Biosciences and Nutrition
>> > Karolinska Institutet
>> > Novum
>> > 14183 Huddinge
>> > Sweden
>> > office: [phone]
>> > LCI room: [phone]
>> > mobile: [phone]
>> >
>> >>
>> >> On 11 Apr 2012, at 22:45, "David Baddeley"
>> >> <[hidden email]>
>> >> wrote:
>> >>
>> >>> *****
>> >>> To join, leave or search the confocal microscopy listserv, go to:
>> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>> *****
>> >>>
>> >>>
>> >>> The diagonal in z will be much 'straighter' (due to the fact that
>> >>> the voxels are
>> >> elongated in z rather than being square), making the factor much
>> >> closer to 1 (probably something like 1.1) so it can safely be
>> >> ignored. When talking about slightly oversampling, 2.3 is already
>> >> doing this - strict Nyquist is a factor of 2. It's also worth
>> >> noting that you should probably use the theoretical resolution
>> >> values (ie
>> >> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU)
>> >> and not the observed PSF width, as these reflect the bandwidth of
>> >> the system. I this tend to reccommend a blanket 70x70x200nm pixel
>> >> size when using a high NA objective on fixed cells. In live cells,
>> >> or other delicate samples you need to exercise a little more
>> >> discretion
>> >> - the artefacts introduced by slight undersampling are likely to
>> >> be
>> outweighed by other considerations.
>> >>>
>> >>> My 2c,
>> >>> David
>> >>>
>> >>>
>> >>> ------------------------------
>> >>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>> >>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Hi John,
>> >>>>
>> >>>> Indirectly, do you suggest the same for Z sampling if we are
>> >>>> interested in 3D measurements?  Thanks
>> >>>>
>> >>>> Monique Vasseur
>> >>>>
>> >>>> -----Message d'origine-----
>> >>>> De : Confocal Microscopy List
>> >> [mailto:[hidden email]] De la part de Lemasters,
>> >> John J.
>> >>>> Envoyé : 11 avril 2012 09:34
>> >>>> À : [hidden email] Objet : Re: Nyquist and
>> >>>> Image size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Please remember that pixel spacing on the diagonal is 1.4 that
>> >>>> in the horizontal
>> >> and vertical directions. Accordingly to meet the Nyquist criterion
>> >> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>> >> Nyquist criterion is an arbitrary threshold, and image quality
>> >> will improve somewhat with sampling greater that proposed by Nyquist.
>> >> Considering diagonal sampling, I suggest using a pixel size that
>> >> is one
>> fourth of the resolving limit for the most critical work.
>> >>>>
>> >>>> John
>> >>>>
>> >>>> --
>> >>>> John J. Lemasters, MD, PhD
>> >>>> Professor and GlaxoSmithKline Distinguished Endowed Chair
>> >>>> Director, Center for Cell Death, Injury & Regeneration
>> >>>> Departments of Pharmaceutical & Biomedical Sciences and
>> >>>> Biochemistry & Molecular Biology Medical University of South
>> >>>> Carolina
>> >>>> DD504 Drug Discovery Building
>> >>>> 70 President Street, MSC 140
>> >>>> Charleston, SC 29425
>> >>>>
>> >>>> Office: 843-876-2360
>> >>>> Lab: 843-876-2354
>> >>>> Fax: [phone]
>> >>>> Email: [hidden email]
>> >>>> http://academicdepartments.musc.edu/ccdir
>> >>>>
>> >>>>
>> >>>> -----Original Message-----
>> >>>> From: Confocal Microscopy List
>> >>>> [mailto:[hidden email]] On Behalf Of John
>> >>>> Oreopoulos
>> >>>> Sent: Wednesday, April 11, 2012 8:29 AM
>> >>>> To: [hidden email]
>> >>>> Subject: Re: Nyquist and Image size
>> >>>>
>> >>>> *****
>> >>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>> *****
>> >>>>
>> >>>> Renato,
>> >>>>
>> >>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>> >>>> Nyquist
>> >> sampling rate (ie: pixel dimensions) does not change since your
>> >> objective lens did not change. The quoted pixel size at 2Kx2K you
>> >> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>> >> (and not gaining anything). Your image may look smoother but it
>> >> contains no more information than the 512x512 image with 90x90 nm
>> >> pixel sizes. Presumably the scan speed is the same between
>> >> 512x512 and 2Kx2K.
>> >>>>
>> >>>> You should decrease the galvometric mirror scan zoom setting to
>> >>>> get back to
>> >> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>> >> Effectively, you will be imaging (and properly sampling) a larger
>> >> field of view then. I'm not familiar with the Leica laser scanning
>> >> confocals so I'm not sure if it will allow you to do this. On
>> >> other systems, like the Olympus FV300 for example, you can set
>> >> your image pixel dimensions (256x256, 512x512, etc.) and your scan
>> >> zoom
>> independently.
>> >>>>
>> >>>> Just out of curiosity, why image 2K x 2K when you can't easily
>> >>>> display that on
>> >> a standard computer screen or present it in a published paper
>> >> without
>> downsizing?
>> >> I rarely departed from 512x512 in my laser scanning days, except
>> >> when I wanted to see a larger field of view.
>> >>>>
>> >>>> Cheers,
>> >>>>
>> >>>>
>> >>>> John Oreopoulos
>> >>>> Research Assistant
>> >>>> Spectral Applied Research
>> >>>> Richmond Hill, Ontario
>> >>>> Canada
>> >>>> www.spectral.ca
>> >>>>
>> >>>>
>> >>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>> >>>>
>> >>>>> *****
>> >>>>> To join, leave or search the confocal microscopy listserv, go to:
>> >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> >>>>> *****
>> >>>>>
>> >>>>> Dear all,
>> >>>>>
>> >>>>> Having attended the first Pawley course in Vancouver I feel
>> >>>>> highly embarassed to ask this, but I would really appreciate a
>> clarification:
>> >>>>>
>> >>>>> When estimating the highest zoom users should apply to their
>> >>>>> sample in order to accommodate for the Nyquist theorem, I
>> >>>>> estimated the optimum pixel size value by dividing the lateral
>> >>>>> resolution (eg: 0.2
>> >>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>> >>>>>
>> >>>>> The doubt: if the image size is increased from 512x512 (having
>> >>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>> >>>>> resulting pixel size (displayed by the system - Leica) the
>> >>>>> pixel size decreases
>> >>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not
>> >>>>> change but only the image size, what happens to Nyquist and the
>> >>>>> optimum pixel size
>> >> at 2Kx2K ?
>> >>>>>
>> >>>>> Many thanks !
>> >>>>>
>> >>>>> Renato
>> >>>>>
>> >>>>> Renato A. Mortara
>> >>>>> Parasitology Division
>> >>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th
>> >>>>> floor São Paulo, SP
>> >>>>> 04023-062
>> >>>>> Brazil
>> >>>>> Phone: [phone]
>> >>>>> Fax:     [phone]
>> >>>>> email: [hidden email]
>> >>>>> home page:  
>> www.ecb.epm.br/~ramortara<http://www.ecb.epm.br/%7Eramortara>
>>
>
>
>
> --
>
>
> Joel B. Sheffield, Ph.D
> Department of Biology
> Temple University
> Philadelphia, PA 19122
> Voice: 215 204 8839
> e-mail: [hidden email]
> URL:  http://astro.temple.edu/~jbs
>
>

[Mark Cannell]

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

That's a nice 'rant' but it does of course ignore the fact that many cameras have square pixels... so it is justified to represent/describe the data with square pixels in that case ... I kind of wish that Microsoft applied similar 'deep thought' to their software before releasing it tho'

LOL

As a further aside,  I note that no one has so far discussed the issue of A/D conversion resolution in deciding the _actual_  bandlimit. For a n bit converter, the bandlimit occurs when the power spectrum of the Airey disk falls below 1/2 a bit (I think) so it's also amplification and noise dependent...

Cheers
Mark


On 14/04/2012, at 11:05 AM, Guy Cox wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> http://alvyray.com/Memos/CG/Microsoft/6_pixel.pdf
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Renato A. Mortara
> Sent: Saturday, 14 April 2012 2:13 AM
> To: [hidden email]
> Subject: Re: Nyquist and Image size
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Guy, it would be great if you could email the article to me:
>
> Thanks again for all the inputs !
>
> Renato
>
>
> Renato A. Mortara
> Disciplina de Parasitologia
> UNIFESP Escola Paulista de Medicina
> R. Botucatu, 862 6o andar
> 04023-062
> São Paulo SP
> Brasil
> [hidden email]
>
>
> Citando "Joel B. Sheffield" <[hidden email]>:
>
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Excellent points.  That paper is a joy to read.
>> Joel
>>
>>
>> On Fri, Apr 13, 2012 at 8:36 AM, Guy Cox <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Well, to put this in more easily understood terms, Nyquist (at 2B)
>>> defines the limit - ie the point where you cease to be able to reconstruct the
>>> wave.   So, as Mark says, you need to get beyond this to actually be able
>>> to get information.  The often-quoted 2.3B more or less corresponds
>>> to the Rayleigh resolution criterion,  ie the point at which you can
>>> reconstruct the wave at usable contrast.  However, the other problem
>>> we face is that we do NOT reconstruct the sine wave, we just look at a map of little squares.
>>> This is stupid.
>>>
>>> Required reading should be:
>>>
>>> A Pixel Is Not A Little Square,
>>> A Pixel Is Not A Little Square,
>>> A Pixel Is Not A Little Square!
>>> (And a Voxel is Not a Little Cube)
>>> Microsoft Technical Memo 6
>>> Alvy Ray Smith
>>> July 17, 1995
>>>
>>> (Yes, that really is the title)
>>>
>>> It's on the Microsoft web site, or I can mail a copy to anyone who is
>>> interested.
>>>
>>>
>>>                   Guy
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List
>>> [mailto:[hidden email]]
>>> On Behalf Of Mark Cannell
>>> Sent: Friday, 13 April 2012 5:53 PM
>>> To: [hidden email]
>>> Subject: Re: Nyquist and Image size
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Please lets not get silly on this. The Nyquist rate is _defined_ as 2
>>> times the bandlimit.  The Nyquist rate is defined by the sufficient
>>> condition for exact reconstructability: Fs > 2B.   2B _is_ the Nyquist rate
>>> as David said, it does not mean Fs = 2B is sufficient!
>>>
>>> Cheers
>>>
>>> On 13/04/2012, at 8:18 AM, Sylvie LeGuyader wrote:
>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> Hi everyone
>>>>
>>>> "strict Nyquist is a factor of 2."
>>>>
>>>> My understanding is that the Nyquist theorem is not arbitrary and
>>>> that
>>> the factor is actually >2. So 2.1 would do as well as 2.3.  If i
>>> understood well the >2 comes from this: if you want to describe a
>>> periodic signal (which is what we do when we acquire an image: we
>>> describe a sum of periodic signals), you need more than 2 points
>>> within 1 full period to collect enough information to reconstruct the periodic signal accurately.
>>> If you only give 2 points per period (e.g. only the crests and
>>> troughs), you can draw the periodic signal is several ways (e.g.
>>> double the frequency of the original signal). When we acquire an
>>> image we should thus sample more than twice the shortest period (the
>>> edges) to acquire enough information for the computer to properly
>>> reconstruct the image. This is why the Nyquist criterion is 'more than 2'. Am I right?
>>>>
>>>> Sylvie
>>>>
>>>> @@@@@@@@@@@@@@@@@@@@@@@@
>>>> Sylvie Le Guyader
>>>> Live Cell Imaging Unit
>>>> Dept of Biosciences and Nutrition
>>>> Karolinska Institutet
>>>> Novum
>>>> 14183 Huddinge
>>>> Sweden
>>>> office: [phone]
>>>> LCI room: [phone]
>>>> mobile: [phone]
>>>>
>>>>>
>>>>> On 11 Apr 2012, at 22:45, "David Baddeley"
>>>>> <[hidden email]>
>>>>> wrote:
>>>>>
>>>>>> *****
>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>> *****
>>>>>>
>>>>>>
>>>>>> The diagonal in z will be much 'straighter' (due to the fact that
>>>>>> the voxels are
>>>>> elongated in z rather than being square), making the factor much
>>>>> closer to 1 (probably something like 1.1) so it can safely be
>>>>> ignored. When talking about slightly oversampling, 2.3 is already
>>>>> doing this - strict Nyquist is a factor of 2. It's also worth
>>>>> noting that you should probably use the theoretical resolution
>>>>> values (ie
>>>>> ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU)
>>>>> and not the observed PSF width, as these reflect the bandwidth of
>>>>> the system. I this tend to reccommend a blanket 70x70x200nm pixel
>>>>> size when using a high NA objective on fixed cells. In live cells,
>>>>> or other delicate samples you need to exercise a little more
>>>>> discretion
>>>>> - the artefacts introduced by slight undersampling are likely to
>>>>> be
>>> outweighed by other considerations.
>>>>>>
>>>>>> My 2c,
>>>>>> David
>>>>>>
>>>>>>
>>>>>> ------------------------------
>>>>>> On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Hi John,
>>>>>>>
>>>>>>> Indirectly, do you suggest the same for Z sampling if we are
>>>>>>> interested in 3D measurements?  Thanks
>>>>>>>
>>>>>>> Monique Vasseur
>>>>>>>
>>>>>>> -----Message d'origine-----
>>>>>>> De : Confocal Microscopy List
>>>>> [mailto:[hidden email]] De la part de Lemasters,
>>>>> John J.
>>>>>>> Envoyé : 11 avril 2012 09:34
>>>>>>> À : [hidden email] Objet : Re: Nyquist and
>>>>>>> Image size
>>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Please remember that pixel spacing on the diagonal is 1.4 that
>>>>>>> in the horizontal
>>>>> and vertical directions. Accordingly to meet the Nyquist criterion
>>>>> for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the
>>>>> Nyquist criterion is an arbitrary threshold, and image quality
>>>>> will improve somewhat with sampling greater that proposed by Nyquist.
>>>>> Considering diagonal sampling, I suggest using a pixel size that
>>>>> is one
>>> fourth of the resolving limit for the most critical work.
>>>>>>>
>>>>>>> John
>>>>>>>
>>>>>>> --
>>>>>>> John J. Lemasters, MD, PhD
>>>>>>> Professor and GlaxoSmithKline Distinguished Endowed Chair
>>>>>>> Director, Center for Cell Death, Injury & Regeneration
>>>>>>> Departments of Pharmaceutical & Biomedical Sciences and
>>>>>>> Biochemistry & Molecular Biology Medical University of South
>>>>>>> Carolina
>>>>>>> DD504 Drug Discovery Building
>>>>>>> 70 President Street, MSC 140
>>>>>>> Charleston, SC 29425
>>>>>>>
>>>>>>> Office: 843-876-2360
>>>>>>> Lab: 843-876-2354
>>>>>>> Fax: [phone]
>>>>>>> Email: [hidden email]
>>>>>>> http://academicdepartments.musc.edu/ccdir
>>>>>>>
>>>>>>>
>>>>>>> -----Original Message-----
>>>>>>> From: Confocal Microscopy List
>>>>>>> [mailto:[hidden email]] On Behalf Of John
>>>>>>> Oreopoulos
>>>>>>> Sent: Wednesday, April 11, 2012 8:29 AM
>>>>>>> To: [hidden email]
>>>>>>> Subject: Re: Nyquist and Image size
>>>>>>>
>>>>>>> *****
>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>> *****
>>>>>>>
>>>>>>> Renato,
>>>>>>>
>>>>>>> Whether you have 256x256, 512x512 or 2048x2048, the "optimum"
>>>>>>> Nyquist
>>>>> sampling rate (ie: pixel dimensions) does not change since your
>>>>> objective lens did not change. The quoted pixel size at 2Kx2K you
>>>>> mentioned (22.5 nm x 22.5 nm) means you are oversampling the image
>>>>> (and not gaining anything). Your image may look smoother but it
>>>>> contains no more information than the 512x512 image with 90x90 nm
>>>>> pixel sizes. Presumably the scan speed is the same between
>>>>> 512x512 and 2Kx2K.
>>>>>>>
>>>>>>> You should decrease the galvometric mirror scan zoom setting to
>>>>>>> get back to
>>>>> an effective pixel size of 90x90 nm with 2Kx2K pixels in your image.
>>>>> Effectively, you will be imaging (and properly sampling) a larger
>>>>> field of view then. I'm not familiar with the Leica laser scanning
>>>>> confocals so I'm not sure if it will allow you to do this. On
>>>>> other systems, like the Olympus FV300 for example, you can set
>>>>> your image pixel dimensions (256x256, 512x512, etc.) and your scan
>>>>> zoom
>>> independently.
>>>>>>>
>>>>>>> Just out of curiosity, why image 2K x 2K when you can't easily
>>>>>>> display that on
>>>>> a standard computer screen or present it in a published paper
>>>>> without
>>> downsizing?
>>>>> I rarely departed from 512x512 in my laser scanning days, except
>>>>> when I wanted to see a larger field of view.
>>>>>>>
>>>>>>> Cheers,
>>>>>>>
>>>>>>>
>>>>>>> John Oreopoulos
>>>>>>> Research Assistant
>>>>>>> Spectral Applied Research
>>>>>>> Richmond Hill, Ontario
>>>>>>> Canada
>>>>>>> www.spectral.ca
>>>>>>>
>>>>>>>
>>>>>>> On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>>>>>>>
>>>>>>>> *****
>>>>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>>>>> *****
>>>>>>>>
>>>>>>>> Dear all,
>>>>>>>>
>>>>>>>> Having attended the first Pawley course in Vancouver I feel
>>>>>>>> highly embarassed to ask this, but I would really appreciate a
>>> clarification:
>>>>>>>>
>>>>>>>> When estimating the highest zoom users should apply to their
>>>>>>>> sample in order to accommodate for the Nyquist theorem, I
>>>>>>>> estimated the optimum pixel size value by dividing the lateral
>>>>>>>> resolution (eg: 0.2
>>>>>>>> microns) by 2.3 so that the value is approxiametely 90 nm.
>>>>>>>>
>>>>>>>> The doubt: if the image size is increased from 512x512 (having
>>>>>>>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the
>>>>>>>> resulting pixel size (displayed by the system - Leica) the
>>>>>>>> pixel size decreases
>>>>>>>> 4 fold, to 22.5 nm. Since the resolution obviously did not
>>>>>>>> change but only the image size, what happens to Nyquist and the
>>>>>>>> optimum pixel size
>>>>> at 2Kx2K ?
>>>>>>>>
>>>>>>>> Many thanks !
>>>>>>>>
>>>>>>>> Renato
>>>>>>>>
>>>>>>>> Renato A. Mortara
>>>>>>>> Parasitology Division
>>>>>>>> UNIFESP - Escola Paulista de Medicina Rua Botucatu, 862, 6th
>>>>>>>> floor São Paulo, SP
>>>>>>>> 04023-062
>>>>>>>> Brazil
>>>>>>>> Phone: [phone]
>>>>>>>> Fax:     [phone]
>>>>>>>> email: [hidden email]
>>>>>>>> home page:  
>>> www.ecb.epm.br/~ramortara<http://www.ecb.epm.br/%7Eramortara>
>>>
>>
>>
>>
>> --
>>
>>
>> Joel B. Sheffield, Ph.D
>> Department of Biology
>> Temple University
>> Philadelphia, PA 19122
>> Voice: 215 204 8839
>> e-mail: [hidden email]
>> URL:  http://astro.temple.edu/~jbs
>>
>>