Optical slice thickness and number for PSF and deconvolution

Tel.: +420 26710 2410

http://www.guycox.com/optical.htm

Re: Optical slice thickness and number for PSF and deconvolution

This is not calculable on the information you supply. In principle, if you are prepared to lose resolution in Z you can use fewer sections and a wider pinhole but this does depend on what your software requires. In the end, the point of doing any sort of deconvolution if you are not sampling at Nyquist is not clear. IMHO, you would be better off working out what sampling your samples can survive and then dealing with the raw data on those terms.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Trnka
Sent: Thursday, 12 August 2010 8:42 PM
To: [hidden email]
Subject: Optical slice thickness and number for PSF and deconvolution

Dear list,

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

Tel.: +420 26710 2410

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00

Vincent Schoonderwoert

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Jan Trnka

*commercial interest*

Dear Jan,

The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.

As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.

In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.

I hope this answers your questions.
Best regards,
Vincent

***********************************************************
Vincent Schoonderwoert, PhD
Scientific Volume Imaging bv
Hilversum, The Netherlands
[hidden email]
[hidden email]
Tel: + 31 35 646 8216
***********************************************************

Jan Trnka wrote:

Dear list,

this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

[hidden email]

Tel.: +420 26710 2410

Jan Trnka

Re: Optical slice thickness and number for PSF and deconvolution

OK, I understand that it's best to sample at Nyquist. However, for my experimental samples (looking at mitochondria in live cells over several hours) this would cause a lot of photodamage and bleaching. Since I don't really care about z-resolution (at this point I basically want a 2D picture of a whole cell with all its mitochondria) I decided to go for a wide pinhole, which allows me to get the picture I want in one go. Maybe this isn't the best way of doing it (any suggestions welcome) but my impression is that such a picture (or a z-stack of thick slices) could be deconvolved to get sharper pictures. Does this make sense? If so, how do I get a PSF for such deconvolution?

Jan

On 12 Aug, 2010, at 2:12 PM, Vincent wrote:

*commercial interest*
Dear Jan,

The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.

As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.

In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.

I hope this answers your questions.
Best regards,
Vincent
***********************************************************
Vincent Schoonderwoert, PhD
Scientific Volume Imaging bv
Hilversum, The Netherlands
[hidden email]
[hidden email]
Tel: + 31 35 646 8216
***********************************************************
Jan Trnka wrote:
Dear list,

this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

[hidden email]

Tel.: +420 26710 2410

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

Tel.: +420 26710 2410

http://www.guycox.com/optical.htm

Re: Optical slice thickness and number for PSF and deconvolution

Well, I’m not clear why you are using confocal at all for this application. If Z resolution doesn’t matter surely you will be better in wide field? And I really can’t see how deconvolution will help. Pace various vendors, if you cannot sample at the resolution limit, I don’t think it will give you any more than conventional noise-reduction and sharpening filters.

Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox CRC Press / Taylor & Francis

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176 Fax +61 2 9351 7682

Mobile 0413 281 861

______________________________________________

http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Trnka
Sent: Thursday, 12 August 2010 10:49 PM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and deconvolution

Jan

On 12 Aug, 2010, at 2:12 PM, Vincent wrote:

*commercial interest*

***********************************************************

Vincent Schoonderwoert, PhD

Scientific Volume Imaging bv

Hilversum, The Netherlands

[hidden email]

[hidden email]

Tel: + 31 35 646 8216

***********************************************************

Jan Trnka wrote:

Dear list,

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

Tel.: +420 26710 2410

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

Tel.: +420 26710 2410

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00

Jan Trnka

Re: Optical slice thickness and number for PSF and deconvolution

<base href="x-msg://117/">Thanks for all the comments. What would be the advantages of using WF over wide-pinhole confocal (assuming it's the same microscope)?

Off list Vincent suggested using 2D deconv. with a theoretical PSF, which might be the way to go. It's my understanding that deconvolution has a place in WF imaging (which I guess is close to what I'm doing), am I correct?

Thanks again,

Jan

On 12 Aug, 2010, at 3:23 PM, Guy Cox wrote:

Well, I’m not clear why you are using confocal at all for this application. If Z resolution doesn’t matter surely you will be better in wide field? And I really can’t see how deconvolution will help.  Pace various vendors, if you cannot sample at the resolution limit, I don’t think it will give you any more than conventional noise-reduction and sharpening filters.

                                                                                        Guy

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

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
______________________________________________
      http://www.guycox.net

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Trnka
Sent: Thursday, 12 August 2010 10:49 PM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and deconvolution

OK, I understand that it's best to sample at Nyquist. However, for my experimental samples (looking at mitochondria in live cells over several hours) this would cause a lot of photodamage and bleaching. Since I don't really care about z-resolution (at this point I basically want a 2D picture of a whole cell with all its mitochondria) I decided to go for a wide pinhole, which allows me to get the picture I want in one go. Maybe this isn't the best way of doing it (any suggestions welcome) but my impression is that such a picture (or a z-stack of thick slices) could be deconvolved to get sharper pictures. Does this make sense? If so, how do I get a PSF for such deconvolution?

Jan

On 12 Aug, 2010, at 2:12 PM, Vincent wrote:

*commercial interest*

Dear Jan,

The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.

As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.

In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.

I hope this answers your questions.
Best regards,
Vincent

***********************************************************
Vincent Schoonderwoert, PhD
Scientific Volume Imaging bv
Hilversum, The Netherlands
[hidden email]
[hidden email]
Tel: + 31 35 646 8216
***********************************************************

Jan Trnka wrote:
Dear list,

this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.

Thanks,

Jan

Jan Trnka, MD, PhD
Department of Biochemistry
3rd Medical Faculty
Ruska 87
100 00 Praha 10
Czech Republic
[hidden email]
Tel.: +420 26710 2410

Jan Trnka, MD, PhD
Department of Biochemistry
3rd Medical Faculty
Ruska 87
100 00 Praha 10
Czech Republic
[hidden email]
Tel.: +420 26710 2410

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

Tel.: +420 26710 2410

Vincent Schoonderwoert

Re: Optical slice thickness and number for PSF and deconvolution

Hi Jan,

Just to make things clear, I would like to add to this that the 2D deconv. is aadvised for WF images.
If you want a single confocal plane, we advise to image at least an adjacent plane on both sides, before performing (3D) deconvolution.

Best regards,
Vincent

Jan Trnka wrote:

<base href="x-msg://117/">Thanks for all the comments. What would be the advantages of using WF over wide-pinhole confocal (assuming it's the same microscope)?

Off list Vincent suggested using 2D deconv. with a theoretical PSF, which might be the way to go. It's my understanding that deconvolution has a place in WF imaging (which I guess is close to what I'm doing), am I correct?

Thanks again,
Jan
On 12 Aug, 2010, at 3:23 PM, Guy Cox wrote:
Well, I’m not clear why you are using confocal at all for this application. If Z resolution doesn’t matter surely you will be better in wide field? And I really can’t see how deconvolution will help.  Pace various vendors, if you cannot sample at the resolution limit, I don’t think it will give you any more than conventional noise-reduction and sharpening filters.

                                                                                        Guy

Optical Imaging Techniques in Cell Biology

by Guy Cox    CRC Press / Taylor & Francis

     http://www.guycox.com/optical.htm

______________________________________________

Associate Professor Guy Cox, MA, DPhil(Oxon)

Australian Centre for Microscopy & Microanalysis,

Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176     Fax +61 2 9351 7682

             Mobile 0413 281 861

______________________________________________

      http://www.guycox.net

From: Confocal Microscopy List [[hidden email]] On Behalf Of Jan Trnka
Sent: Thursday, 12 August 2010 10:49 PM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and deconvolution

OK, I understand that it's best to sample at Nyquist. However, for my experimental samples (looking at mitochondria in live cells over several hours) this would cause a lot of photodamage and bleaching. Since I don't really care about z-resolution (at this point I basically want a 2D picture of a whole cell with all its mitochondria) I decided to go for a wide pinhole, which allows me to get the picture I want in one go. Maybe this isn't the best way of doing it (any suggestions welcome) but my impression is that such a picture (or a z-stack of thick slices) could be deconvolved to get sharper pictures. Does this make sense? If so, how do I get a PSF for such deconvolution?

Jan

On 12 Aug, 2010, at 2:12 PM, Vincent wrote:

*commercial interest*

Dear Jan,

The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.

As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.

In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.

I hope this answers your questions.
Best regards,
Vincent

***********************************************************

Vincent Schoonderwoert, PhD

Scientific Volume Imaging bv

Hilversum, The Netherlands

[hidden email]

[hidden email]

Tel: + 31 35 646 8216

***********************************************************

Jan Trnka wrote:

Dear list,

this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.

Thanks,

Jan

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

[hidden email]

Tel.: +420 26710 2410

Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

[hidden email]

Tel.: +420 26710 2410

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00
Jan Trnka, MD, PhD

Department of Biochemistry

3rd Medical Faculty

Ruska 87

100 00 Praha 10

Czech Republic

[hidden email]

Tel.: +420 26710 2410

-- 
***********************************************************
Vincent Schoonderwoert, PhD
Imaging Specialist/Account Manager
[hidden email]

Scientific Volume Imaging bv
Laapersveld 63
1213 VB Hilversum, The Netherlands
Tel: + 31 35 646 8216
Fax: + 31 35 683 7971
www.svi.nl
[hidden email]
***********************************************************
SVI Customer support: mail us your questions [hidden email]
or find answers online in our FAQ: http://support.svi.nl

Mario-2

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Jan Trnka

Jan,

The back and forth about your problem seems to have muddied things a bit.

About the first question below, WF systems generally speaking have
much higher light throughput and more efficient light sensors for a
higher overall quantum detection efficiency. Thus, less exposure for
the same S/N is usually available using WF. It is not a question of
simply using the same microscope; it is the entire light path that it
is at issue. Only way to be certain comparing two systems is to use a
robust sample (no photobleaching, e.g., fast decay time phosphor
grains).

Deconvolution is a requirement for standard WF 3D imaging. It more
than has a place, it is essential.

If you are limited to a confocal and you need more signal, opening
your pinhole and applying deconvolution is reasonable. For whatever
pinhole size you choose, you will need to acquire a 3D PSF. I believe
you first asked what PSF to use; opinions will vary but my preference
is to acquire bead/reference object PSF data appropriate to the
objective and wavelengths you are using; at minimum, match the
Nyquist criterion in x,y,z. From the reference data for each
objective estimate the theoretical 3D PSF function and use that to
deconvolve your images. (I prefer using the theoretical since it
won't directly add noise to the decon. process)

To actually get any benefit from deconvolution you have to sample
your specimen using Nyquist spacing. It doesn't matter whether you
are using WF or large pinholes. Except for speed, 2D deconvolution is
practically worthless (sorry I am being a little harsh); it is no
better than an Unsharp filter. Actually, using a Fourier filter and
"notching out the very lowest frequencies" does a pretty good job of
removing haze and greatly improves contrast, and it is quite fast.

Mario

>Thanks for all the comments. What would be the advantages of using
>WF over wide-pinhole confocal (assuming it's the same microscope)?
>
>Off list Vincent suggested using 2D deconv. with a theoretical PSF,
>which might be the way to go. It's my understanding that
>deconvolution has a place in WF imaging (which I guess is close to
>what I'm doing), am I correct?
>
>Thanks again,
>
>Jan
>
>....

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Vincent Schoonderwoert

Hi All

I'm sorry but this advice is wrong. The pinhole is a control that
_should_ be used when decreased (mainly z) resolution is acceptable. The
lasers can then be turned down and, if desired, decon. can be used to
help clean up the image. The problem is that many users want a "pretty
picture" but pretty pictures may not be needed for quantification of
(say) number of mitochondria. As we say on the Vancouver course, "Every
photon is precious" and you may also increase signal by accepting a
wider spectral band or using an LP filter. The key to good experimental
work is to understand what measurement you want and then to pick
conditions that allow you to get sufficient data with sufficient (not
too many) time points to answer your question. Do you need a full 3D
image or will a couple of slices suffice? Use a high NA lens. As others
have said, consider using widefield with a high QE CCD if you really
don't need the maximum possible resolution in 3D...

My 2c

Mark Cannell

Vincent wrote:

> *commercial interest*
>
>
> Dear Jan,
>
> The amount of the signal in images is mostly judged just after image
> acquisition. Based on this it is often decided to use a wider pinhole.
> As you probably know, when deconvolution is properly performed you will gain not
> only an increase in resolution but also in signal. Therefore, we advise to close
> the pinhole and use deconvolution for increasing the signal (to noise) before
> determining the quality of the image.
>
> As with imaging the object of interest it is important to follow the Nyquist
> criteria for imaging the bead images.
> We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to
> determine these rates. You can also create a picture here of your theoretical
> PSF to get an idea of its dimensions.
>
> In general it is best to really match the Nyquist criterion in xyz. Else you can
> go for 2x more. This however may introduce other problems like e.g., bleaching.
> If the bead images are differently sampled it requires interpolation for
> matching that, making the process of deconvolution more computationally
> demanding. Thus Nyquist is okay. Another important thing to keep in mind is that
> you need to image enough planes to cover your PSF.
>
> I hope this answers your questions.
> Best regards,
> Vincent
>
> ***********************************************************
> Vincent Schoonderwoert, PhD
> Scientific Volume Imaging bv
> Hilversum, The Netherlands
> [hidden email]
> [hidden email]
> Tel: + 31 35 646 8216
> ***********************************************************
>
>
>
>
>
> Jan Trnka wrote:
> > Dear list,
> >
> > this is probably a trivial question but so far I haven't found a good answer.
> > When taking 3D images of subresolution beads in a confocal microscope (for PSF
> > construction) does the number and thickness of slices in the z-stack need to
> > be exactly the same as that of a sample to be deconvolved? I understand the
> > x-y dimensions need to be the same but how does it work for z? Would a higher
> > number of thinner slices (finer z resolution) of the bead improve the
> > construction of the PSF? My actual samples are imaged with a rather wide
> > pinhole setting to limit the exposure of the sample (live cells) and thus
> > provide quite thick optical sections.
> >
> > Thanks,
> >
> > Jan
> >
> > Jan Trnka, MD, PhD
> > Department of Biochemistry
> > 3rd Medical Faculty
> > Ruska 87
> > 100 00 Praha 10
> > Czech Republic
> > [hidden email] <mailto:[hidden email]>
> > Tel.: +420 26710 2410
> >
> >
> >
>
>
>
>

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Jan Trnka

Deconvolution can be applied to any imaging process. In 2D you will not
control for out of focus light, but noise will decrease.

Hope this helps.

Mark

Jan Trnka wrote:

> Thanks for all the comments. What would be the advantages of using WF over wide-pinhole confocal (assuming it's the same microscope)?
>
> Off list Vincent suggested using 2D deconv. with a theoretical PSF, which might be the way to go. It's my understanding that deconvolution has a place in WF imaging (which I guess is close to what I'm doing), am I correct?
>
> Thanks again,
>
> Jan
>
> On 12 Aug, 2010, at 3:23 PM, Guy Cox wrote:
>
>
>> Well, I’m not clear why you are using confocal at all for this application. If Z resolution doesn’t matter surely you will be better in wide field? And I really can’t see how deconvolution will help. Pace various vendors, if you cannot sample at the resolution limit, I don’t think it will give you any more than conventional noise-reduction and sharpening filters.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor & Francis
>> http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________________________
>> http://www.guycox.net
>>
>>
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jan Trnka
>> Sent: Thursday, 12 August 2010 10:49 PM
>> To: [hidden email]
>> Subject: Re: Optical slice thickness and number for PSF and deconvolution
>>
>> OK, I understand that it's best to sample at Nyquist. However, for my experimental samples (looking at mitochondria in live cells over several hours) this would cause a lot of photodamage and bleaching. Since I don't really care about z-resolution (at this point I basically want a 2D picture of a whole cell with all its mitochondria) I decided to go for a wide pinhole, which allows me to get the picture I want in one go. Maybe this isn't the best way of doing it (any suggestions welcome) but my impression is that such a picture (or a z-stack of thick slices) could be deconvolved to get sharper pictures. Does this make sense? If so, how do I get a PSF for such deconvolution?
>>
>> Jan
>>
>> On 12 Aug, 2010, at 2:12 PM, Vincent wrote:
>>
>>
>> *commercial interest*
>>
>> Dear Jan,
>>
>> The amount of the signal in images is mostly judged just after image acquisition. Based on this it is often decided to use a wider pinhole.
>> As you probably know, when deconvolution is properly performed you will gain not only an increase in resolution but also in signal. Therefore, we advise to close the pinhole and use deconvolution for increasing the signal (to noise) before determining the quality of the image.
>>
>> As with imaging the object of interest it is important to follow the Nyquist criteria for imaging the bead images.
>> We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to determine these rates. You can also create a picture here of your theoretical PSF to get an idea of its dimensions.
>>
>> In general it is best to really match the Nyquist criterion in xyz. Else you can go for 2x more. This however may introduce other problems like e.g., bleaching. If the bead images are differently sampled it requires interpolation for matching that, making the process of deconvolution more computationally demanding. Thus Nyquist is okay. Another important thing to keep in mind is that you need to image enough planes to cover your PSF.
>>
>> I hope this answers your questions.
>> Best regards,
>> Vincent
>>
>> ***********************************************************
>> Vincent Schoonderwoert, PhD
>> Scientific Volume Imaging bv
>> Hilversum, The Netherlands
>> [hidden email]
>> [hidden email]
>> Tel: + 31 35 646 8216
>> ***********************************************************
>>
>>
>>
>>
>> Jan Trnka wrote:
>> Dear list,
>>
>> this is probably a trivial question but so far I haven't found a good answer. When taking 3D images of subresolution beads in a confocal microscope (for PSF construction) does the number and thickness of slices in the z-stack need to be exactly the same as that of a sample to be deconvolved? I understand the x-y dimensions need to be the same but how does it work for z? Would a higher number of thinner slices (finer z resolution) of the bead improve the construction of the PSF? My actual samples are imaged with a rather wide pinhole setting to limit the exposure of the sample (live cells) and thus provide quite thick optical sections.
>>
>> Thanks,
>>
>> Jan
>>
>> Jan Trnka, MD, PhD
>> Department of Biochemistry
>> 3rd Medical Faculty
>> Ruska 87
>> 100 00 Praha 10
>> Czech Republic
>> [hidden email]
>> Tel.: +420 26710 2410
>>
>>
>>
>>
>>
>>
>>
>>
>> Jan Trnka, MD, PhD
>> Department of Biochemistry
>> 3rd Medical Faculty
>> Ruska 87
>> 100 00 Praha 10
>> Czech Republic
>> [hidden email]
>> Tel.: +420 26710 2410
>>
>>
>>
>> No virus found in this incoming message.
>> Checked by AVG - www.avg.com
>> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00
>>
>>
>
> Jan Trnka, MD, PhD
> Department of Biochemistry
> 3rd Medical Faculty
> Ruska 87
> 100 00 Praha 10
> Czech Republic
> [hidden email]
> Tel.: +420 26710 2410
>
>
>
>
>

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Vincent Schoonderwoert

Dear Vincent,

I disagree, taking 3 planes will not permit calculation of a single
plane in 3D unless there is no signal outside the measured planes! Just
3 planes is too sparse when signal originates from outside the plane of
interest. Taking the 2 extra planes can be used to reduce blur (i.e.
increase contrast) but is is not deconvolution.

Hope this helps

Mark Cannell

Vincent wrote:

> Hi Jan,
>
> Just to make things clear, I would like to add to this that the 2D deconv. is
> aadvised for WF images.
> If you want a single confocal plane, we advise to image at least an adjacent
> plane on both sides, before performing (3D) deconvolution.
>
> Best regards,
> Vincent
>
>
>
> Jan Trnka wrote:
> > Thanks for all the comments. What would be the advantages of using WF over
> > wide-pinhole confocal (assuming it's the same microscope)?
> >
> > Off list Vincent suggested using 2D deconv. with a theoretical PSF, which
> > might be the way to go. It's my understanding that deconvolution has a place
> > in WF imaging (which I guess is close to what I'm doing), am I correct?
> >
> > Thanks again,
> >
> > Jan
> >
> > On 12 Aug, 2010, at 3:23 PM, Guy Cox wrote:
> >
> >> Well, I’m not clear why you are using confocal at all for this application.
> >> If Z resolution doesn’t matter surely you will be better in wide field? And
> >> I really can’t see how deconvolution will help. /Pace/ various vendors, if
> >> you cannot sample at the resolution limit, I don’t think it will give you any
> >> more than conventional noise-reduction and sharpening filters.
> >>
> >>
> >> Guy
> >>
> >> Optical Imaging Techniques in Cell Biology
> >> by Guy Cox CRC Press / Taylor & Francis
> >> http://www.guycox.com/optical.htm
> >> ______________________________________________
> >> Associate Professor Guy Cox, MA, DPhil(Oxon)
> >> Australian Centre for Microscopy & Microanalysis,
> >> Madsen Building F09, University of Sydney, NSW 2006
> >>
> >> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> >> Mobile 0413 281 861
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> *From:* Confocal Microscopy List
> >> [mailto:[hidden email]] *On Behalf Of *Jan Trnka
> >> *Sent:* Thursday, 12 August 2010 10:49 PM
> >> *To:* [hidden email] <mailto:[hidden email]>
> >> *Subject:* Re: Optical slice thickness and number for PSF and deconvolution
> >>
> >> OK, I understand that it's best to sample at Nyquist. However, for my
> >> experimental samples (looking at mitochondria in live cells over several
> >> hours) this would cause a lot of photodamage and bleaching. Since I don't
> >> really care about z-resolution (at this point I basically want a 2D picture
> >> of a whole cell with all its mitochondria) I decided to go for a wide
> >> pinhole, which allows me to get the picture I want in one go. Maybe this
> >> isn't the best way of doing it (any suggestions welcome) but my impression is
> >> that such a picture (or a z-stack of thick slices) could be deconvolved to
> >> get sharper pictures. Does this make sense? If so, how do I get a PSF for
> >> such deconvolution?
> >>
> >> Jan
> >>
> >> On 12 Aug, 2010, at 2:12 PM, Vincent wrote:
> >>
> >>
> >> *commercial interest*
> >>
> >> Dear Jan,
> >>
> >> The amount of the signal in images is mostly judged just after image
> >> acquisition. Based on this it is often decided to use a wider pinhole.
> >> As you probably know, when deconvolution is properly performed you will gain
> >> not only an increase in resolution but also in signal. Therefore, we advise
> >> to close the pinhole and use deconvolution for increasing the signal (to
> >> noise) before determining the quality of the image.
> >>
> >> As with imaging the object of interest it is important to follow the Nyquist
> >> criteria for imaging the bead images.
> >> We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator
> >> <http://www.svi.nl/NyquistCalculator>) to determine these rates. You can also
> >> create a picture here of your theoretical PSF to get an idea of its dimensions.
> >>
> >> In general it is best to really match the Nyquist criterion in xyz. Else you
> >> can go for 2x more. This however may introduce other problems like e.g.,
> >> bleaching. If the bead images are differently sampled it requires
> >> interpolation for matching that, making the process of deconvolution more
> >> computationally demanding. Thus Nyquist is okay. Another important thing to
> >> keep in mind is that you need to image enough planes to cover your PSF.
> >>
> >> I hope this answers your questions.
> >> Best regards,
> >> Vincent
> >>
> >> ***********************************************************
> >> Vincent Schoonderwoert, PhD
> >> Scientific Volume Imaging bv
> >> Hilversum, The Netherlands
> >> [hidden email] <mailto:[hidden email]>
> >> [hidden email] <mailto:[hidden email]>
> >> Tel: + 31 35 646 8216
> >> ***********************************************************
> >>
> >>
> >>
> >>
> >> Jan Trnka wrote:
> >> Dear list,
> >>
> >> this is probably a trivial question but so far I haven't found a good answer.
> >> When taking 3D images of subresolution beads in a confocal microscope (for
> >> PSF construction) does the number and thickness of slices in the z-stack need
> >> to be exactly the same as that of a sample to be deconvolved? I understand
> >> the x-y dimensions need to be the same but how does it work for z? Would a
> >> higher number of thinner slices (finer z resolution) of the bead improve the
> >> construction of the PSF? My actual samples are imaged with a rather wide
> >> pinhole setting to limit the exposure of the sample (live cells) and thus
> >> provide quite thick optical sections.
> >>
> >> Thanks,
> >>
> >> Jan
> >>
> >> Jan Trnka, MD, PhD
> >> Department of Biochemistry
> >> 3rd Medical Faculty
> >> Ruska 87
> >> 100 00 Praha 10
> >> Czech Republic
> >> [hidden email] <mailto:[hidden email]>
> >> Tel.: +420 26710 2410
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >> Jan Trnka, MD, PhD
> >> Department of Biochemistry
> >> 3rd Medical Faculty
> >> Ruska 87
> >> 100 00 Praha 10
> >> Czech Republic
> >> [hidden email] <mailto:[hidden email]>
> >> Tel.: +420 26710 2410
> >>
> >>
> >>
> >>
> >> No virus found in this incoming message.
> >> Checked by AVG - www.avg.com <http://www.avg.com>
> >> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10 04:34:00
> >>
> >
> > Jan Trnka, MD, PhD
> > Department of Biochemistry
> > 3rd Medical Faculty
> > Ruska 87
> > 100 00 Praha 10
> > Czech Republic
> > [hidden email] <mailto:[hidden email]>
> > Tel.: +420 26710 2410
> >
> >
> >
>
>
> --
> ***********************************************************
> Vincent Schoonderwoert, PhD
> Imaging Specialist/Account Manager
> [hidden email]
>
> Scientific Volume Imaging bv
> Laapersveld 63
> 1213 VB Hilversum, The Netherlands
> Tel: + 31 35 646 8216
> Fax: + 31 35 683 7971
> www.svi.nl
> [hidden email]
> ***********************************************************
> SVI Customer support: mail us your questions [hidden email]
> or find answers online in our FAQ: http://support.svi.nl
>
>
>
>

Alison J. North

Position available: Light Microscopy Research Support Specialist in the Rockefe ller University=?windows-1252?Q?=92s?= Bio-Imaging Resource Center

In reply to this post by Mario-2

/Please note – this is a different position from the one I posted two
days ago – it’s my bad luck that two of my excellent staff members are
moving to other parts of the country around the same time!/

The Rockefeller University is seeking a qualified and motivated
full-time microscopist for its Bio-Imaging Resource Center. The
Research Support Specialist will assist in advising and training users
in all areas of optical microscopy, including microscope set-up,
experimental design, sample preparation and data evaluation. The
Specialist will also perform trouble-shooting, microscope maintenance
and miscellaneous laboratory maintenance and job-related duties as
required and will report to the Director of the Center. Collaborative
studies with RU researchers will also be encouraged for motivated
individuals.

The Bio-Imaging Resource Center is used by around 70 laboratories at RU
and nearby institutions and we enjoy strong support within the
University. Equipment in the center currently includes a new Olympus
FV1000 MPE Twin upright multiphoton system, a Zeiss LSM 510 inverted
multiphoton system, two Zeiss LSM 510 confocal microscopes (one with
META detector), two DeltaVision image restoration microscopes (including
lasers for FRAP and photoactivation), a Yokogawa-type spinning disk
confocal microscope fitted with the Photonics Instruments Digital Mosaic
system for FRAP/Photoactivation, a Nikon/Lambert Instruments multi-line
TIRF/FLIM system, an MMI laser microdissection system, and various
widefield fluorescence microscopes. We hope to add superresolution
microscopy capabilities over the next few months.

Master’s degree or Ph.D. in biology or a related field and a minimum of
two years of experience in advanced fluorescence microscopy, preferably
involving live cell imaging, plus experience with sample preparation and
image analysis.* *Must have excellent interpersonal and communication
skills, an enthusiastic approach towards new techniques, motivation to
maintain a broad knowledge of state-of-the-art imaging technology and
the flexibility to interact with a diverse group of researchers. Strong
organizational and multi-tasking skills are also essential.

The Rockefeller University is located on a beautiful campus on
Manhattan's Upper East Side. We offer a competitive salary,
comprehensive benefits and an excellent working environment.

To apply for this position, click the following URL, click on 'staff
opportunities" and enter keyword "IRC9002":

http://www.rockefeller.edu/hr/career.php

For specific technical questions about the BIRC’s instrumentation or
services, please feel free to contact Dr. Alison North, the Director of
the BIRC, at: [hidden email] <mailto:[hidden email]>

/The Rockefeller University is an Affirmative Action/Equal Employment
Opportunity/VEVRAA employer./

--
Alison J. North, Ph.D.,
Research Assistant Professor and
Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab ++ 212 327 7486
Fax: ++ 212 327 7489

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Mark Cannell

It's a bit challenging to disagree with Mark but .... we are interested
in signal over noise. Opening the pinhole beyond the diameter of the
Airy disk will let in a little more signal (from the outer rings) and a
LOT more noise. In almost every case it will make things worse.
Photons are precious - if they come from where we want - other photons
are something we need to exclude at all costs.

As to the question about 3 sections - Mark is quite right, of course, if
we are dealing with a thick sample, but if I've followed this thread
correctly we are dealing with thin cells where the information is
largely in one plane. If this is so we should be able to do pretty good
deconvolution with 3 sections.

Guy

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

Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Mark Cannell
Sent: Friday, 13 August 2010 8:52 AM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and
deconvolution

Hi All

I'm sorry but this advice is wrong. The pinhole is a control that
_should_ be used when decreased (mainly z) resolution is acceptable. The

lasers can then be turned down and, if desired, decon. can be used to
help clean up the image. The problem is that many users want a "pretty
picture" but pretty pictures may not be needed for quantification of
(say) number of mitochondria. As we say on the Vancouver course, "Every

photon is precious" and you may also increase signal by accepting a
wider spectral band or using an LP filter. The key to good experimental

work is to understand what measurement you want and then to pick
conditions that allow you to get sufficient data with sufficient (not
too many) time points to answer your question. Do you need a full 3D
image or will a couple of slices suffice? Use a high NA lens. As others
have said, consider using widefield with a high QE CCD if you really
don't need the maximum possible resolution in 3D...

My 2c

Mark Cannell

Vincent wrote:
> *commercial interest*
>
>
> Dear Jan,
>
> The amount of the signal in images is mostly judged just after image
> acquisition. Based on this it is often decided to use a wider pinhole.
> As you probably know, when deconvolution is properly performed you
will gain not
> only an increase in resolution but also in signal. Therefore, we
advise to close
> the pinhole and use deconvolution for increasing the signal (to noise)
before
> determining the quality of the image.
>
> As with imaging the object of interest it is important to follow the
Nyquist
> criteria for imaging the bead images.
> We have a Nyquist calculator on our website
(www.svi.nl/NyquistCalculator) to
> determine these rates. You can also create a picture here of your
theoretical
> PSF to get an idea of its dimensions.
>
> In general it is best to really match the Nyquist criterion in xyz.
Else you can
> go for 2x more. This however may introduce other problems like e.g.,
bleaching.
> If the bead images are differently sampled it requires interpolation
for
> matching that, making the process of deconvolution more
computationally
> demanding. Thus Nyquist is okay. Another important thing to keep in
mind is that

> you need to image enough planes to cover your PSF.
>
> I hope this answers your questions.
> Best regards,
> Vincent
>
> ***********************************************************
> Vincent Schoonderwoert, PhD
> Scientific Volume Imaging bv
> Hilversum, The Netherlands
> [hidden email]
> [hidden email]
> Tel: + 31 35 646 8216
> ***********************************************************
>
>
>
>
>
> Jan Trnka wrote:
> > Dear list,
> >
> > this is probably a trivial question but so far I haven't found a

good answer.
> > When taking 3D images of subresolution beads in a confocal
microscope (for PSF
> > construction) does the number and thickness of slices in the z-stack
need to
> > be exactly the same as that of a sample to be deconvolved? I
understand the
> > x-y dimensions need to be the same but how does it work for z? Would
a higher
> > number of thinner slices (finer z resolution) of the bead improve
the
> > construction of the PSF? My actual samples are imaged with a rather
wide
> > pinhole setting to limit the exposure of the sample (live cells) and
thus

> > provide quite thick optical sections.
> >
> > Thanks,
> >
> > Jan
> >
> > Jan Trnka, MD, PhD
> > Department of Biochemistry
> > 3rd Medical Faculty
> > Ruska 87
> > 100 00 Praha 10
> > Czech Republic
> > [hidden email] <mailto:[hidden email]>
> > Tel.: +420 26710 2410
> >
> >
> >
>
>
>
>

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
04:34:00

Sudipta Maiti

Re: Optical slice thickness and number for PSF and deconvolution

Wait a minute. Since Mark and Guy are involved, there is something useful to
be learned here, and I was reading this with intent. But the original
question had two points that I feel were not adequately addressed by either.
First, the number of Z-slices required for imaging the bead: more than the
original can help, as the deconvolution algorithm will probably use a smooth
function to model the PSF obtained from the subresolution bead image, and use
that for deconvolution.
Second, was there something about using a larger pinhole during the actual
image acquisition? The pinhole size should match for the actual imaging and
the bead - otherwise you don't get the same PSF. I guess it is possible to
calculte the PSF for other pinhole sizes, but may not be the best thing to do.
Sudipta

On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote

> It's a bit challenging to disagree with Mark but .... we are interested
> in signal over noise. Opening the pinhole beyond the diameter of the
> Airy disk will let in a little more signal (from the outer rings)
> and a LOT more noise. In almost every case it will make things worse.
> Photons are precious - if they come from where we want - other
> photons are something we need to exclude at all costs.
>
> As to the question about 3 sections - Mark is quite right, of course,
> if we are dealing with a thick sample, but if I've followed this thread
> correctly we are dealing with thin cells where the information is
> largely in one plane. If this is so we should be able to do pretty good
> deconvolution with 3 sections.
>
> Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
> http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> Mobile 0413 281 861
> ______________________________________________
> http://www.guycox.net
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: Friday, 13 August 2010 8:52 AM
> To: [hidden email]
> Subject: Re: Optical slice thickness and number for PSF and
> deconvolution
>
> Hi All
>
> I'm sorry but this advice is wrong. The pinhole is a control that
> _should_ be used when decreased (mainly z) resolution is acceptable.
> The
>
> lasers can then be turned down and, if desired, decon. can be used
> to help clean up the image. The problem is that many users want a
> "pretty picture" but pretty pictures may not be needed for
> quantification of
> (say) number of mitochondria. As we say on the Vancouver course, "Every
>
> photon is precious" and you may also increase signal by accepting a
> wider spectral band or using an LP filter. The key to good experimental
>
> work is to understand what measurement you want and then to pick
> conditions that allow you to get sufficient data with sufficient
> (not too many) time points to answer your question. Do you need a
> full 3D image or will a couple of slices suffice? Use a high NA
> lens. As others have said, consider using widefield with a high QE
> CCD if you really don't need the maximum possible resolution in 3D...
>
> My 2c
>
> Mark Cannell
>
> Vincent wrote:
> > *commercial interest*
> >
> >
> > Dear Jan,
> >
> > The amount of the signal in images is mostly judged just after image
> > acquisition. Based on this it is often decided to use a wider pinhole.
> > As you probably know, when deconvolution is properly performed you
> will gain not
> > only an increase in resolution but also in signal. Therefore, we
> advise to close
> > the pinhole and use deconvolution for increasing the signal (to noise)
> before
> > determining the quality of the image.
> >
> > As with imaging the object of interest it is important to follow the
> Nyquist
> > criteria for imaging the bead images.
> > We have a Nyquist calculator on our website
> (www.svi.nl/NyquistCalculator) to
> > determine these rates. You can also create a picture here of your
> theoretical
> > PSF to get an idea of its dimensions.
> >
> > In general it is best to really match the Nyquist criterion in xyz.
> Else you can
> > go for 2x more. This however may introduce other problems like e.g.,
> bleaching.
> > If the bead images are differently sampled it requires interpolation
> for
> > matching that, making the process of deconvolution more
> computationally
> > demanding. Thus Nyquist is okay. Another important thing to keep in
> mind is that
> > you need to image enough planes to cover your PSF.
> >
> > I hope this answers your questions.
> > Best regards,
> > Vincent
> >
> > ***********************************************************
> > Vincent Schoonderwoert, PhD
> > Scientific Volume Imaging bv
> > Hilversum, The Netherlands
> > [hidden email]
> > [hidden email]
> > Tel: + 31 35 646 8216
> > ***********************************************************
> >
> >
> >
> >
> >
> > Jan Trnka wrote:
> > > Dear list,
> > >
> > > this is probably a trivial question but so far I haven't found a
> good answer.
> > > When taking 3D images of subresolution beads in a confocal
> microscope (for PSF
> > > construction) does the number and thickness of slices in the z-stack
> need to
> > > be exactly the same as that of a sample to be deconvolved? I
> understand the
> > > x-y dimensions need to be the same but how does it work for z? Would
> a higher
> > > number of thinner slices (finer z resolution) of the bead improve
> the
> > > construction of the PSF? My actual samples are imaged with a rather
> wide
> > > pinhole setting to limit the exposure of the sample (live cells) and
> thus
> > > provide quite thick optical sections.
> > >
> > > Thanks,
> > >
> > > Jan
> > >
> > > Jan Trnka, MD, PhD
> > > Department of Biochemistry
> > > 3rd Medical Faculty
> > > Ruska 87
> > > 100 00 Praha 10
> > > Czech Republic
> > > [hidden email] <mailto:[hidden email]>
> > > Tel.: +420 26710 2410
> > >
> > >
> > >
> >
> >
> >
> >
>
> No virus found in this incoming message.
> Checked by AVG - www.avg.com
> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
> 04:34:00

Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
alternate e-mail: [hidden email]
url: biophotonics.wetpaint.com

mmodel

Re: Optical slice thickness and number for PSF and deconvolution

Dear Jan,

I absolutely agree with Sudipta: (1) use the same pinhole to measure PSF as you use for imaging, (2) extra resolution in acquiring the PSF won't hurt.
There are many other considerations, of course, for example: PSF at the edge of the field is usually wider than in the center; if the cells are thick, then spherical aberration may strongly affect the PSF at deeper layers, and you might get better results by using a theoretical PSF that takes spherical aberration into account than with experimental PSF; it also helps to remember about axial scaling...

Mike Model

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Sudipta Maiti [[hidden email]]
Sent: Saturday, August 14, 2010 12:36 PM
To: [hidden email]
Subject: Re: Optical slice thickness and number for PSF and deconvolution

Wait a minute. Since Mark and Guy are involved, there is something useful to
be learned here, and I was reading this with intent. But the original
question had two points that I feel were not adequately addressed by either.
First, the number of Z-slices required for imaging the bead: more than the
original can help, as the deconvolution algorithm will probably use a smooth
function to model the PSF obtained from the subresolution bead image, and use
that for deconvolution.
Second, was there something about using a larger pinhole during the actual
image acquisition? The pinhole size should match for the actual imaging and
the bead - otherwise you don't get the same PSF. I guess it is possible to
calculte the PSF for other pinhole sizes, but may not be the best thing to do.
Sudipta

On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Sudipta Maiti

Hi Guy

I'm going to have to disagree with some of what you said because we are
talking about live cell imaging. As I'm sure we all know, opening the
pinhole has a big effect on z resolution and that is the main reason why
signal goes up in 3D objects (adding the wings of the in plane Airy disk
alone only adds about 25% signal). It's the increase in the size of the
sampled volume that generates more signal. Now I can see that you think
of an out of focus object as a "noise" generator, and in that sense I
agree with you, but if we are talking about thin cells how many bright
well out of focus objects are there? I really don't think you can say
that opening the pinhole will let in a LOT more "noise "(your definition
here) unless you know what the object really is. There is always a trade
off between S/N and resolution, so if you can take a reduction in
resolution you will be able to turn the lasers down and that is what
this thread started with -keeping cells alive.

I'm quite inflexible in my view on this, the correct _live cell_
solution is to open the pinhole until the x,y,z resolution is as low as
you can accept and then get the needed S/N with minimum laser power
-this is what we teach on the live cell course in Vancouver -it's all
about the keeping the cell healthy while getting just enough information
to answer the experimental question (and not minimizing "noise" per se.).

Yes, I agree with you that if the object z extent is << PSFz, you have
enough information to get a reasonable solution to the convolution
problem. But in that special case I suggest you don't need a confocal at
all! For very thin objects a WF microscope with a good CCD (+/-
deconvolution) will give a better result than a confocal (even with the
pinhole wide open as the confocal detector/optics are less efficient).

But in most other cases, a confocal is used to reject significant out of
focus light (as you seem acknowledge in your description of "noise"
increasing with pinhole diameter) and that contribution cannot be
properly defined by just 3 planes within the PSF (that's sometimes
called "nearest neighbours deconvolution" -but it's not a correct
solution to the deconvolution problem). Now you may use some simple
software to take the 3 planes of data to increase contrast and reduce
noise in the center plane but the image is not _deconvolved_ at all but
rather "deblurred" I think this is the case because it is not possible
to _know_ that all _out of focus_ information has been correctly
estimated with just 3 planes -and that is because of _photon noise_.

For an intuitive approach I like to think of it this way, if 3
planes(=samples) can only supply enough information to solve the spatial
problem (2 for Nyquist plus 1 for outside plane of focus generated
signal), what extra information is available to identify/control noise?
If you sketch a problem as 1D on a piece of paper one can see how all
the information in 3 planes is used for the spatial problem (e.g. if the
object is A.sin(wx)+B you have 3 unknowns A, B, w so you need 3
samples). Now if you add Poisson noise so that you know that the mean is
equal to the variance, how do you get an estimate of the mean when all
the variance in your 3 samples is already assigned to the equation
variables you just solved? From this I think it's clear why noise
introduces a problem and kills the 3 plane deconvolution solution for an
extended object. Let me be quite clear here, having extra information
planes can always help improve the in plane image (I call it
"deblurring" or "denoising" depending on the intent/method of the
processing) but it is not possible to actually deconvolve until
sufficient information has been captured to solve the spatial problem
_and_ get some estimate of noise. On the other hand, if the problem is
so constrained that you _know_ there is no data outside the plane of
focus (so that you have all the information needed to solve the
noise/spatial problem with 3 planes -and on this I think we agree) then
you shouldn't be using a confocal anyway IMO.

Cheers Mark

> On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote
>
>> It's a bit challenging to disagree with Mark but .... we are interested
>> in signal over noise. Opening the pinhole beyond the diameter of the
>> Airy disk will let in a little more signal (from the outer rings)
>> and a LOT more noise. In almost every case it will make things worse.
>> Photons are precious - if they come from where we want - other
>> photons are something we need to exclude at all costs.
>>
>> As to the question about 3 sections - Mark is quite right, of course,
>> if we are dealing with a thick sample, but if I've followed this thread
>> correctly we are dealing with thin cells where the information is
>> largely in one plane. If this is so we should be able to do pretty good
>> deconvolution with 3 sections.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor & Francis
>> http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________________________
>> http://www.guycox.net
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 August 2010 8:52 AM
>> To: [hidden email]
>> Subject: Re: Optical slice thickness and number for PSF and
>> deconvolution
>>
>> Hi All
>>
>> I'm sorry but this advice is wrong. The pinhole is a control that
>> _should_ be used when decreased (mainly z) resolution is acceptable.
>> The
>>
>> lasers can then be turned down and, if desired, decon. can be used
>> to help clean up the image. The problem is that many users want a
>> "pretty picture" but pretty pictures may not be needed for
>> quantification of
>> (say) number of mitochondria. As we say on the Vancouver course, "Every
>>
>> photon is precious" and you may also increase signal by accepting a
>> wider spectral band or using an LP filter. The key to good experimental
>>
>> work is to understand what measurement you want and then to pick
>> conditions that allow you to get sufficient data with sufficient
>> (not too many) time points to answer your question. Do you need a
>> full 3D image or will a couple of slices suffice? Use a high NA
>> lens. As others have said, consider using widefield with a high QE
>> CCD if you really don't need the maximum possible resolution in 3D...
>>
>> My 2c
>>
>> Mark Cannell
>>
>> Vincent wrote:
>>
>>> *commercial interest*
>>>
>>>
>>> Dear Jan,
>>>
>>> The amount of the signal in images is mostly judged just after image
>>> acquisition. Based on this it is often decided to use a wider pinhole.
>>> As you probably know, when deconvolution is properly performed you
>>>
>> will gain not
>>
>>> only an increase in resolution but also in signal. Therefore, we
>>>
>> advise to close
>>
>>> the pinhole and use deconvolution for increasing the signal (to noise)
>>>
>> before
>>
>>> determining the quality of the image.
>>>
>>> As with imaging the object of interest it is important to follow the
>>>
>> Nyquist
>>
>>> criteria for imaging the bead images.
>>> We have a Nyquist calculator on our website
>>>
>> (www.svi.nl/NyquistCalculator) to
>>
>>> determine these rates. You can also create a picture here of your
>>>
>> theoretical
>>
>>> PSF to get an idea of its dimensions.
>>>
>>> In general it is best to really match the Nyquist criterion in xyz.
>>>
>> Else you can
>>
>>> go for 2x more. This however may introduce other problems like e.g.,
>>>
>> bleaching.
>>
>>> If the bead images are differently sampled it requires interpolation
>>>
>> for
>>
>>> matching that, making the process of deconvolution more
>>>
>> computationally
>>
>>> demanding. Thus Nyquist is okay. Another important thing to keep in
>>>
>> mind is that
>>
>>> you need to image enough planes to cover your PSF.
>>>
>>> I hope this answers your questions.
>>> Best regards,
>>> Vincent
>>>
>>> ***********************************************************
>>> Vincent Schoonderwoert, PhD
>>> Scientific Volume Imaging bv
>>> Hilversum, The Netherlands
>>> [hidden email]
>>> [hidden email]
>>> Tel: + 31 35 646 8216
>>> ***********************************************************
>>>
>>>
>>>
>>>
>>>
>>> Jan Trnka wrote:
>>>
>>>> Dear list,
>>>>
>>>> this is probably a trivial question but so far I haven't found a
>>>>
>> good answer.
>>
>>>> When taking 3D images of subresolution beads in a confocal
>>>>
>> microscope (for PSF
>>
>>>> construction) does the number and thickness of slices in the z-stack
>>>>
>> need to
>>
>>>> be exactly the same as that of a sample to be deconvolved? I
>>>>
>> understand the
>>
>>>> x-y dimensions need to be the same but how does it work for z? Would
>>>>
>> a higher
>>
>>>> number of thinner slices (finer z resolution) of the bead improve
>>>>
>> the
>>
>>>> construction of the PSF? My actual samples are imaged with a rather
>>>>
>> wide
>>
>>>> pinhole setting to limit the exposure of the sample (live cells) and
>>>>
>> thus
>>
>>>> provide quite thick optical sections.
>>>>
>>>> Thanks,
>>>>
>>>> Jan
>>>>
>>>> Jan Trnka, MD, PhD
>>>> Department of Biochemistry
>>>> 3rd Medical Faculty
>>>> Ruska 87
>>>> 100 00 Praha 10
>>>> Czech Republic
>>>> [hidden email] <mailto:[hidden email]>
>>>> Tel.: +420 26710 2410
>>>>
>>>>
>>>>
>>>>
>>>
>>>
>>>
>> No virus found in this incoming message.
>> Checked by AVG - www.avg.com
>> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
>> 04:34:00
>>
>
>
> Dr. Sudipta Maiti
> Associate Professor
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: 91-22-2280-4610
> alternate e-mail: [hidden email]
> url: biophotonics.wetpaint.com
>

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by mmodel

Quite so, you need to know the PSF that exists in the _sample_. So if
you open the pinhole to keep the cell alive or even use an oil lens in
water (shudders) that is what you need to do to your beads too. That
said, if the object is noise reduction and not resolution, you may not
be interested in 3D at all, and in that case you case use just 2D
deconvolution to help control noise.

Hope this helps.

Mark

> ar Jan,
>
> I absolutely agree with Sudipta: (1) use the same pinhole to measure PSF as you use for imaging, (2) extra resolution in acquiring the PSF won't hurt.
> There are many other considerations, of course, for example: PSF at the edge of the field is usually wider than in the center; if the cells are thick, then spherical aberration may strongly affect the PSF at deeper layers, and you might get better results by using a theoretical PSF that takes spherical aberration into account than with experimental PSF; it also helps to remember about axial scaling...
>
> Mike Model
>
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On Behalf Of Sudipta Maiti [[hidden email]]
> Sent: Saturday, August 14, 2010 12:36 PM
> To: [hidden email]
> Subject: Re: Optical slice thickness and number for PSF and deconvolution
>
> Wait a minute. Since Mark and Guy are involved, there is something useful to
> be learned here, and I was reading this with intent. But the original
> question had two points that I feel were not adequately addressed by either.
> First, the number of Z-slices required for imaging the bead: more than the
> original can help, as the deconvolution algorithm will probably use a smooth
> function to model the PSF obtained from the subresolution bead image, and use
> that for deconvolution.
> Second, was there something about using a larger pinhole during the actual
> image acquisition? The pinhole size should match for the actual imaging and
> the bead - otherwise you don't get the same PSF. I guess it is possible to
> calculte the PSF for other pinhole sizes, but may not be the best thing to do.
> Sudipta
>
> On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote
>
>> It's a bit challenging to disagree with Mark but .... we are interested
>> in signal over noise. Opening the pinhole beyond the diameter of the
>> Airy disk will let in a little more signal (from the outer rings)
>> and a LOT more noise. In almost every case it will make things worse.
>> Photons are precious - if they come from where we want - other
>> photons are something we need to exclude at all costs.
>>
>> As to the question about 3 sections - Mark is quite right, of course,
>> if we are dealing with a thick sample, but if I've followed this thread
>> correctly we are dealing with thin cells where the information is
>> largely in one plane. If this is so we should be able to do pretty good
>> deconvolution with 3 sections.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor & Francis
>> http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________________________
>> http://www.guycox.net
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 August 2010 8:52 AM
>> To: [hidden email]
>> Subject: Re: Optical slice thickness and number for PSF and
>> deconvolution
>>
>> Hi All
>>
>> I'm sorry but this advice is wrong. The pinhole is a control that
>> _should_ be used when decreased (mainly z) resolution is acceptable.
>> The
>>
>> lasers can then be turned down and, if desired, decon. can be used
>> to help clean up the image. The problem is that many users want a
>> "pretty picture" but pretty pictures may not be needed for
>> quantification of
>> (say) number of mitochondria. As we say on the Vancouver course, "Every
>>
>> photon is precious" and you may also increase signal by accepting a
>> wider spectral band or using an LP filter. The key to good experimental
>>
>> work is to understand what measurement you want and then to pick
>> conditions that allow you to get sufficient data with sufficient
>> (not too many) time points to answer your question. Do you need a
>> full 3D image or will a couple of slices suffice? Use a high NA
>> lens. As others have said, consider using widefield with a high QE
>> CCD if you really don't need the maximum possible resolution in 3D...
>>
>> My 2c
>>
>> Mark Cannell
>>
>> Vincent wrote:
>>
>>> *commercial interest*
>>>
>>>
>>> Dear Jan,
>>>
>>> The amount of the signal in images is mostly judged just after image
>>> acquisition. Based on this it is often decided to use a wider pinhole.
>>> As you probably know, when deconvolution is properly performed you
>>>
>> will gain not
>>
>>> only an increase in resolution but also in signal. Therefore, we
>>>
>> advise to close
>>
>>> the pinhole and use deconvolution for increasing the signal (to noise)
>>>
>> before
>>
>>> determining the quality of the image.
>>>
>>> As with imaging the object of interest it is important to follow the
>>>
>> Nyquist
>>
>>> criteria for imaging the bead images.
>>> We have a Nyquist calculator on our website
>>>
>> (www.svi.nl/NyquistCalculator) to
>>
>>> determine these rates. You can also create a picture here of your
>>>
>> theoretical
>>
>>> PSF to get an idea of its dimensions.
>>>
>>> In general it is best to really match the Nyquist criterion in xyz.
>>>
>> Else you can
>>
>>> go for 2x more. This however may introduce other problems like e.g.,
>>>
>> bleaching.
>>
>>> If the bead images are differently sampled it requires interpolation
>>>
>> for
>>
>>> matching that, making the process of deconvolution more
>>>
>> computationally
>>
>>> demanding. Thus Nyquist is okay. Another important thing to keep in
>>>
>> mind is that
>>
>>> you need to image enough planes to cover your PSF.
>>>
>>> I hope this answers your questions.
>>> Best regards,
>>> Vincent
>>>
>>> ***********************************************************
>>> Vincent Schoonderwoert, PhD
>>> Scientific Volume Imaging bv
>>> Hilversum, The Netherlands
>>> [hidden email]
>>> [hidden email]
>>> Tel: + 31 35 646 8216
>>> ***********************************************************
>>>
>>>
>>>
>>>
>>>
>>> Jan Trnka wrote:
>>>
>>>> Dear list,
>>>>
>>>> this is probably a trivial question but so far I haven't found a
>>>>
>> good answer.
>>
>>>> When taking 3D images of subresolution beads in a confocal
>>>>
>> microscope (for PSF
>>
>>>> construction) does the number and thickness of slices in the z-stack
>>>>
>> need to
>>
>>>> be exactly the same as that of a sample to be deconvolved? I
>>>>
>> understand the
>>
>>>> x-y dimensions need to be the same but how does it work for z? Would
>>>>
>> a higher
>>
>>>> number of thinner slices (finer z resolution) of the bead improve
>>>>
>> the
>>
>>>> construction of the PSF? My actual samples are imaged with a rather
>>>>
>> wide
>>
>>>> pinhole setting to limit the exposure of the sample (live cells) and
>>>>
>> thus
>>
>>>> provide quite thick optical sections.
>>>>
>>>> Thanks,
>>>>
>>>> Jan
>>>>
>>>> Jan Trnka, MD, PhD
>>>> Department of Biochemistry
>>>> 3rd Medical Faculty
>>>> Ruska 87
>>>> 100 00 Praha 10
>>>> Czech Republic
>>>> [hidden email] <mailto:[hidden email]>
>>>> Tel.: +420 26710 2410
>>>>
>>>>
>>>>
>>>>
>>>
>>>
>>>
>> No virus found in this incoming message.
>> Checked by AVG - www.avg.com
>> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
>> 04:34:00
>>
>
>
> Dr. Sudipta Maiti
> Associate Professor
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: 91-22-2280-4610
> alternate e-mail: [hidden email]
> url: biophotonics.wetpaint.com

Hans

Re: Optical slice thickness and number for PSF and deconvolution

In reply to this post by Jan Trnka

Dear Sudipta and All,

(Message from a commercial vendor)

On 08/14/2010 06:36 PM, Sudipta Maiti wrote:
> Wait a minute. Since Mark and Guy are involved, there is something useful to
> be learned here, and I was reading this with intent. But the original
> question had two points that I feel were not adequately addressed by either.
Good point!
> First, the number of Z-slices required for imaging the bead: more than the

To record a bead or multi-bead image to derive a PSF from it, it should
be recorded at the Nyquist rate or better. The number of Z-planes should
be sufficient to fully contain the PSF. In a typical confocal case 30+
planes should do it. There are a number of caveats though, see for example:

http://www.svi.nl/RecordingBeads
http://www.svi.nl/PsfFromBeads&highlight=distiller

In short, recording the bead is quite independent of recording the
biological object, provided that optical conditions are the same. If
bleaching or time constraints dictate few planes of the object, then
from the standpoint of deconvolution a slight undersampling in Z is
acceptable. But not too much!

> original can help, as the deconvolution algorithm will probably use a smooth
> function to model the PSF obtained from the subresolution bead image, and use
> that for deconvolution.
> Second, was there something about using a larger pinhole during the actual
> image acquisition? The pinhole size should match for the actual imaging and
> the bead - otherwise you don't get the same PSF. I guess it is possible to
> calculte the PSF for other pinhole sizes, but may not be the best thing to do.
Indeed, the pinhole sizes used in recording the PSF and the data should
match. The sampling can be adapted and the tails of the PSF can be
extrapolated though it is better if this is not necessary.

Regarding the tradeoff between pinhole size, microscope type, signal and
deconvolution, the outcome of that is very much object dependent. As a
rule of thumb, with small sparse objects you're likely to be better off
with a WF system + deconvolution, with thick dense objects with a
confocal system. Increasing the pinhole beyond the Airy disk size gets
you less Z-resolution and more signal in the absolute sense, but as Guy
pointed out more (blurry) signal from adjacent areas carries also more
noise. Deconvolution can repair the resolution loss, again much
depending on the sparseness of the object. For example, if there are
strongly labelled horizontal membranes in your image next to other
interesting but faint features, opening the pinhole is probably not a
good idea.

Regarding the number of planes in the data to be recorded for
deconvolution: if possible record enough planes to contain the entire
object. If that is not feasible, then the more the better.
In the low noise WF sparse object case even single plane deconvolution
is possible (this is not 2D deconvolution!), in the confocal case three
or more Nyquist sampled planes are needed. Of course reliability is not
as good that of a full-object deconvolution, and there are cases where
3-plane confocal 'deconvolution' will fail. But as long as the object is
fairly sparse and there are no large bright objects right outside the
recorded volume, my colleague Vincent's point was that chances are good.
The catch is this: because the volume in the data contributing to a
deconvolved pixel is less in the 3-plane case compared to a full-object
deconvolution, the signal to noise (SNR) requirements are a bit higher
than in the full object case. If the choice is between 3 higher SNR
planes or more lower SNR planes, both cases involving the same amount of
detected photons, best go for more planes.

We've done simulations on this topic in the past, if anyone is
interested we could construct a wiki page.

-- Hans

> Sudipta
>
> On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote
>> It's a bit challenging to disagree with Mark but .... we are interested
>> in signal over noise. Opening the pinhole beyond the diameter of the
>> Airy disk will let in a little more signal (from the outer rings)
>> and a LOT more noise. In almost every case it will make things worse.
>> Photons are precious - if they come from where we want - other
>> photons are something we need to exclude at all costs.
>>
>> As to the question about 3 sections - Mark is quite right, of course,
>> if we are dealing with a thick sample, but if I've followed this thread
>> correctly we are dealing with thin cells where the information is
>> largely in one plane. If this is so we should be able to do pretty good
>> deconvolution with 3 sections.
>>
>> Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox CRC Press / Taylor& Francis
>> http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy& Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone +61 2 9351 3176 Fax +61 2 9351 7682
>> Mobile 0413 281 861
>> ______________________________________________
>> http://www.guycox.net
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 August 2010 8:52 AM
>> To: [hidden email]
>> Subject: Re: Optical slice thickness and number for PSF and
>> deconvolution
>>
>> Hi All
>>
>> I'm sorry but this advice is wrong. The pinhole is a control that
>> _should_ be used when decreased (mainly z) resolution is acceptable.
>> The
>>
>> lasers can then be turned down and, if desired, decon. can be used
>> to help clean up the image. The problem is that many users want a
>> "pretty picture" but pretty pictures may not be needed for
>> quantification of
>> (say) number of mitochondria. As we say on the Vancouver course, "Every
>>
>> photon is precious" and you may also increase signal by accepting a
>> wider spectral band or using an LP filter. The key to good experimental
>>
>> work is to understand what measurement you want and then to pick
>> conditions that allow you to get sufficient data with sufficient
>> (not too many) time points to answer your question. Do you need a
>> full 3D image or will a couple of slices suffice? Use a high NA
>> lens. As others have said, consider using widefield with a high QE
>> CCD if you really don't need the maximum possible resolution in 3D...
>>
>> My 2c
>>
>> Mark Cannell
>>
>> Vincent wrote:
>>> *commercial interest*
>>>
>>>
>>> Dear Jan,
>>>
>>> The amount of the signal in images is mostly judged just after image
>>> acquisition. Based on this it is often decided to use a wider pinhole.
>>> As you probably know, when deconvolution is properly performed you
>> will gain not
>>> only an increase in resolution but also in signal. Therefore, we
>> advise to close
>>> the pinhole and use deconvolution for increasing the signal (to noise)
>> before
>>> determining the quality of the image.
>>>
>>> As with imaging the object of interest it is important to follow the
>> Nyquist
>>> criteria for imaging the bead images.
>>> We have a Nyquist calculator on our website
>> (www.svi.nl/NyquistCalculator) to
>>> determine these rates. You can also create a picture here of your
>> theoretical
>>> PSF to get an idea of its dimensions.
>>>
>>> In general it is best to really match the Nyquist criterion in xyz.
>> Else you can
>>> go for 2x more. This however may introduce other problems like e.g.,
>> bleaching.
>>> If the bead images are differently sampled it requires interpolation
>> for
>>> matching that, making the process of deconvolution more
>> computationally
>>> demanding. Thus Nyquist is okay. Another important thing to keep in
>> mind is that
>>> you need to image enough planes to cover your PSF.
>>>
>>> I hope this answers your questions.
>>> Best regards,
>>> Vincent
>>>
>>> ***********************************************************
>>> Vincent Schoonderwoert, PhD
>>> Scientific Volume Imaging bv
>>> Hilversum, The Netherlands
>>> [hidden email]
>>> [hidden email]
>>> Tel: + 31 35 646 8216
>>> ***********************************************************
>>>
>>>
>>>
>>>
>>>
>>> Jan Trnka wrote:
>>>> Dear list,
>>>>
>>>> this is probably a trivial question but so far I haven't found a
>> good answer.
>>>> When taking 3D images of subresolution beads in a confocal
>> microscope (for PSF
>>>> construction) does the number and thickness of slices in the z-stack
>> need to
>>>> be exactly the same as that of a sample to be deconvolved? I
>> understand the
>>>> x-y dimensions need to be the same but how does it work for z? Would
>> a higher
>>>> number of thinner slices (finer z resolution) of the bead improve
>> the
>>>> construction of the PSF? My actual samples are imaged with a rather
>> wide
>>>> pinhole setting to limit the exposure of the sample (live cells) and
>> thus
>>>> provide quite thick optical sections.
>>>>
>>>> Thanks,
>>>>
>>>> Jan
>>>>
>>>> Jan Trnka, MD, PhD
>>>> Department of Biochemistry
>>>> 3rd Medical Faculty
>>>> Ruska 87
>>>> 100 00 Praha 10
>>>> Czech Republic
>>>> [hidden email]<mailto:[hidden email]>
>>>> Tel.: +420 26710 2410
>>>>
>>>>
>>>>
>>>
>>>
>>>
>>>
>>
>> No virus found in this incoming message.
>> Checked by AVG - www.avg.com
>> Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
>> 04:34:00
>
>
> Dr. Sudipta Maiti
> Associate Professor
> Dept. of Chemical Sciences
> Tata Institute of Fundamental Research
> Homi Bhabha Raod, Colaba, Mumbai 400005
> Ph. 91-22-2278-2716 / 2539
> Fax: 91-22-2280-4610
> alternate e-mail: [hidden email]
> url: biophotonics.wetpaint.com
>

--
-------------------------------------------------------------------
dr. Hans T.M. van der Voort ([hidden email])
Scientific Volume Imaging b.v., URL: http://www.svi.nl/

Mario-2

Re: Optical slice thickness and number for PSF and deconvolution

Dear listers,

Could someone let me know if they received my post from Thurs. past
concerning the optical slice issue. For this topic, I claim no
special insight that hasn't been repeated a number of times by
subsequent posts. It is just that more than a few times my
submissions have been completely ignored. Are others seeing my
submissions or not? If someone could let me know off list or on I
would appreciate it. Thanks,

Mario

>Dear Sudipta and All,
>
>(Message from a commercial vendor)
>
>On 08/14/2010 06:36 PM, Sudipta Maiti wrote:
>>Wait a minute. Since Mark and Guy are involved, there is something useful to
>>be learned here, and I was reading this with intent. But the original
>>question had two points that I feel were not adequately addressed by either.
>Good point!
>>First, the number of Z-slices required for imaging the bead: more than the
>
>To record a bead or multi-bead image to derive a PSF from it, it should
>be recorded at the Nyquist rate or better. The number of Z-planes should
>be sufficient to fully contain the PSF. In a typical confocal case 30+
>planes should do it. There are a number of caveats though, see for example:
>
>http://www.svi.nl/RecordingBeads
>http://www.svi.nl/PsfFromBeads&highlight=distiller
>
>In short, recording the bead is quite independent of recording the
>biological object, provided that optical conditions are the same. If
>bleaching or time constraints dictate few planes of the object, then
>from the standpoint of deconvolution a slight undersampling in Z is
>acceptable. But not too much!
>
>>original can help, as the deconvolution algorithm will probably use a smooth
>>function to model the PSF obtained from the subresolution bead image, and use
>>that for deconvolution.
>>Second, was there something about using a larger pinhole during the actual
>>image acquisition? The pinhole size should match for the actual imaging and
>>the bead - otherwise you don't get the same PSF. I guess it is possible to
>>calculte the PSF for other pinhole sizes, but may not be the best
>>thing to do.
>Indeed, the pinhole sizes used in recording the PSF and the data should
>match. The sampling can be adapted and the tails of the PSF can be
>extrapolated though it is better if this is not necessary.
>
>Regarding the tradeoff between pinhole size, microscope type, signal and
>deconvolution, the outcome of that is very much object dependent. As a
>rule of thumb, with small sparse objects you're likely to be better off
>with a WF system + deconvolution, with thick dense objects with a
>confocal system. Increasing the pinhole beyond the Airy disk size gets
>you less Z-resolution and more signal in the absolute sense, but as Guy
>pointed out more (blurry) signal from adjacent areas carries also more
>noise. Deconvolution can repair the resolution loss, again much
>depending on the sparseness of the object. For example, if there are
>strongly labelled horizontal membranes in your image next to other
>interesting but faint features, opening the pinhole is probably not a
>good idea.
>
>Regarding the number of planes in the data to be recorded for
>deconvolution: if possible record enough planes to contain the entire
>object. If that is not feasible, then the more the better.
>In the low noise WF sparse object case even single plane deconvolution
>is possible (this is not 2D deconvolution!), in the confocal case three
>or more Nyquist sampled planes are needed. Of course reliability is not
>as good that of a full-object deconvolution, and there are cases where
>3-plane confocal 'deconvolution' will fail. But as long as the object is
>fairly sparse and there are no large bright objects right outside the
>recorded volume, my colleague Vincent's point was that chances are good.
>The catch is this: because the volume in the data contributing to a
>deconvolved pixel is less in the 3-plane case compared to a full-object
>deconvolution, the signal to noise (SNR) requirements are a bit higher
>than in the full object case. If the choice is between 3 higher SNR
>planes or more lower SNR planes, both cases involving the same amount of
>detected photons, best go for more planes.
>
>We've done simulations on this topic in the past, if anyone is
>interested we could construct a wiki page.
>
>-- Hans
>
>>Sudipta
>>
>> On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote
>>>It's a bit challenging to disagree with Mark but .... we are interested
>>>in signal over noise. Opening the pinhole beyond the diameter of the
>>>Airy disk will let in a little more signal (from the outer rings)
>>> and a LOT more noise. In almost every case it will make things worse.
>>>Photons are precious - if they come from where we want - other
>>>photons are something we need to exclude at all costs.
>>>
>>>As to the question about 3 sections - Mark is quite right, of course,
>>> if we are dealing with a thick sample, but if I've followed this thread
>>>correctly we are dealing with thin cells where the information is
>>>largely in one plane. If this is so we should be able to do pretty good
>>>deconvolution with 3 sections.
>>>
>>> Guy
>>>
>>>Optical Imaging Techniques in Cell Biology
>>>by Guy Cox CRC Press / Taylor& Francis
>>> http://www.guycox.com/optical.htm
>>>______________________________________________
>>>Associate Professor Guy Cox, MA, DPhil(Oxon)
>>>Australian Centre for Microscopy& Microanalysis,
>>>Madsen Building F09, University of Sydney, NSW 2006
>>>
>>>Phone +61 2 9351 3176 Fax +61 2 9351 7682
>>> Mobile 0413 281 861
>>>______________________________________________
>>> http://www.guycox.net
>>>
>>>-----Original Message-----
>>>From: Confocal Microscopy List [mailto:[hidden email]]
>>>On Behalf Of Mark Cannell
>>>Sent: Friday, 13 August 2010 8:52 AM
>>>To: [hidden email]
>>>Subject: Re: Optical slice thickness and number for PSF and
>>>deconvolution
>>>
>>>Hi All
>>>
>>>I'm sorry but this advice is wrong. The pinhole is a control that
>>>_should_ be used when decreased (mainly z) resolution is acceptable.
>>>The
>>>
>>>lasers can then be turned down and, if desired, decon. can be used
>>>to help clean up the image. The problem is that many users want a
>>>"pretty picture" but pretty pictures may not be needed for
>>>quantification of
>>>(say) number of mitochondria. As we say on the Vancouver course, "Every
>>>
>>>photon is precious" and you may also increase signal by accepting a
>>>wider spectral band or using an LP filter. The key to good experimental
>>>
>>>work is to understand what measurement you want and then to pick
>>>conditions that allow you to get sufficient data with sufficient
>>>(not too many) time points to answer your question. Do you need a
>>>full 3D image or will a couple of slices suffice? Use a high NA
>>>lens. As others have said, consider using widefield with a high QE
>>>CCD if you really don't need the maximum possible resolution in 3D...
>>>
>>>My 2c
>>>
>>>Mark Cannell
>>>
>>>Vincent wrote:
>>>>*commercial interest*
>>>>
>>>>
>>>>Dear Jan,
>>>>
>>>>The amount of the signal in images is mostly judged just after image
>>>>acquisition. Based on this it is often decided to use a wider pinhole.
>>>>As you probably know, when deconvolution is properly performed you
>>>will gain not
>>>>only an increase in resolution but also in signal. Therefore, we
>>>advise to close
>>>>the pinhole and use deconvolution for increasing the signal (to noise)
>>>before
>>>>determining the quality of the image.
>>>>
>>>>As with imaging the object of interest it is important to follow the
>>>Nyquist
>>>>criteria for imaging the bead images.
>>>>We have a Nyquist calculator on our website
>>>(www.svi.nl/NyquistCalculator) to
>>>>determine these rates. You can also create a picture here of your
>>>theoretical
>>>>PSF to get an idea of its dimensions.
>>>>
>>>>In general it is best to really match the Nyquist criterion in xyz.
>>>Else you can
>>>>go for 2x more. This however may introduce other problems like e.g.,
>>>bleaching.
>>>>If the bead images are differently sampled it requires interpolation
>>>for
>>>>matching that, making the process of deconvolution more
>>>computationally
>>>>demanding. Thus Nyquist is okay. Another important thing to keep in
>>>mind is that
>>>>you need to image enough planes to cover your PSF.
>>>>
>>>>I hope this answers your questions.
>>>>Best regards,
>>>>Vincent
>>>>
>>>>***********************************************************
>>>>Vincent Schoonderwoert, PhD
>>>>Scientific Volume Imaging bv
>>>>Hilversum, The Netherlands
>>>>[hidden email]
>>>>[hidden email]
>>>>Tel: + 31 35 646 8216
>>>>***********************************************************
>>>>
>>>>
>>>>
>>>>
>>>>
>>>>Jan Trnka wrote:
>>>>>Dear list,
>>>>>
>>>>>this is probably a trivial question but so far I haven't found a
>>>good answer.
>>>>>When taking 3D images of subresolution beads in a confocal
>>>microscope (for PSF
>>>>>construction) does the number and thickness of slices in the z-stack
>>>need to
>>>>>be exactly the same as that of a sample to be deconvolved? I
>>>understand the
>>>>>x-y dimensions need to be the same but how does it work for z? Would
>>>a higher
>>>>>number of thinner slices (finer z resolution) of the bead improve
>>>the
>>>>>construction of the PSF? My actual samples are imaged with a rather
>>>wide
>>>>>pinhole setting to limit the exposure of the sample (live cells) and
>>>thus
>>>>>provide quite thick optical sections.
>>>>>
>>>>>Thanks,
>>>>>
>>>>>Jan
>>>>>
>>>>>Jan Trnka, MD, PhD
>>>>>Department of Biochemistry
>>>>>3rd Medical Faculty
>>>>>Ruska 87
>>>>>100 00 Praha 10
>>>>>Czech Republic
>>>>>[hidden email]<mailto:[hidden email]>
>>>>>Tel.: +420 26710 2410
>>>>>
>>>>>
>>>>
>>>>
>>>>
>>>>
>>>
>>>No virus found in this incoming message.
>>>Checked by AVG - www.avg.com
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>>
>>
>>Dr. Sudipta Maiti
>>Associate Professor
>>Dept. of Chemical Sciences
>>Tata Institute of Fundamental Research
>>Homi Bhabha Raod, Colaba, Mumbai 400005
>>Ph. 91-22-2278-2716 / 2539
>>Fax: 91-22-2280-4610
>>alternate e-mail: [hidden email]
>>url: biophotonics.wetpaint.com
>>
>
>
>--
>-------------------------------------------------------------------
>dr. Hans T.M. van der Voort ([hidden email])
>Scientific Volume Imaging b.v., URL: http://www.svi.nl/

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]