Re: Deconvolution Algorithms Continued

[neeraj Gohad-3]

I would like to thank everyone who replied to the Deconvolution Algorithm questions. Once I Get back to the lab on Monday we  will start comparing the suggested algorithms. Thanks Brian for your comments as well, the commercial product we are comparing against is the Autoquant module in the Nikon NIS elements software.

 

 I do have a question for all of you regarding calculating the point spread function.  For some of the open source algorithms we are using we have to input the PSF in the deconvolution algorithm.  For this we using a an open source software which calculates theoretical PSF after you input imaging parameters such as, magnification, NA etc. but this software does not have Laser Scanning Confocal microscopy as an imaging modality option, it only has wide field fluorescence. Is there a formula for theoretically  calculating PSF in which you can input  the Confocal pin hole as a parameter, or would it be easier to determine the PSF experimentally using fluorescent beads?

 

Best Regards,

 

Neeraj.

 

Neeraj V. Gohad, PhD

Postdoctoral Fellow,

Okeanos Research Group

Department of Biological Sciences

132 Long Hall,Clemson University

Clemson, SC-29634

864-656-3597

[hidden email]

 

[Lutz Schaefer]

Neeraj,

 

a confocal PSF (or generally, any incoherent PSF of an aplanatic microscope system) is the product of the excitation and emission distributions. The exitation PSF could be approximated by a widefield PSF and the emission PSF by a widefield PSF (using the emission wavelength) but convolved by the pinhole geometry. You need to be careful with sampling and scaling issues. Other than that its not a big deal. The approximation with widefield vs point-scanner had been okay in nearly all cases that came across my work. For the calculation of widefield PSF's I suggest the classic Sarah Gibson and Frad Lanni's paper "Experimental test of an analytical model of aberration in an oil immersion objective lens used in three dimensional light microscopy" JOSA V.9 N.1 1992 (which was to the best of my knowledge also implemented in XCOSM). But of course, you may also use the slightly more accurate vector wave model.

 

I hope this was of some help

Lutz

 

____________________________________
Lutz Schaefer
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, Ontario, N2P 2A2
Phone/Fax: [phone]
Email:     [hidden email]
Website: http://home.golden.net/~lschafer/
____________________________________

 

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

From: [hidden email]

To: [hidden email]

Sent: Saturday, November 29, 2008 5:25 PM

Subject: Re: Deconvolution Algorithms Continued

I would like to thank everyone who replied to the Deconvolution Algorithm questions. Once I Get back to the lab on Monday we  will start comparing the suggested algorithms. Thanks Brian for your comments as well, the commercial product we are comparing against is the Autoquant module in the Nikon NIS elements software.

 

 I do have a question for all of you regarding calculating the point spread function.  For some of the open source algorithms we are using we have to input the PSF in the deconvolution algorithm.  For this we using a an open source software which calculates theoretical PSF after you input imaging parameters such as, magnification, NA etc. but this software does not have Laser Scanning Confocal microscopy as an imaging modality option, it only has wide field fluorescence. Is there a formula for theoretically  calculating PSF in which you can input  the Confocal pin hole as a parameter, or would it be easier to determine the PSF experimentally using fluorescent beads?

 

Best Regards,

 

Neeraj.

 

Neeraj V. Gohad, PhD

Postdoctoral Fellow,

Okeanos Research Group

Department of Biological Sciences

132 Long Hall,Clemson University

Clemson, SC-29634

864-656-3597

[hidden email]

 

[Northan, Brian]

 

A correction to my previous post, I had mistakenly said the latest version of Autoquant was X2.3, the latest release is actually X2.1.1.   There will be an X2.1.3 released in the next few weeks.

 

Brian

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Lutz Schaefer
Sent: Saturday, November 29, 2008 5:55 PM
To: [hidden email]
Subject: Re: Deconvolution Algorithms Continued

 

Neeraj,

 

a confocal PSF (or generally, any incoherent PSF of an aplanatic microscope system) is the product of the excitation and emission distributions. The exitation PSF could be approximated by a widefield PSF and the emission PSF by a widefield PSF (using the emission wavelength) but convolved by the pinhole geometry. You need to be careful with sampling and scaling issues. Other than that its not a big deal. The approximation with widefield vs point-scanner had been okay in nearly all cases that came across my work. For the calculation of widefield PSF's I suggest the classic Sarah Gibson and Frad Lanni's paper "Experimental test of an analytical model of aberration in an oil immersion objective lens used in three dimensional light microscopy" JOSA V.9 N.1 1992 (which was to the best of my knowledge also implemented in XCOSM). But of course, you may also use the slightly more accurate vector wave model.

 

I hope this was of some help

Lutz

 

____________________________________
Lutz Schaefer
Advanced Imaging Methodology Consultation
16-715 Doon Village Rd.
Kitchener, Ontario, N2P 2A2
Phone/Fax: [phone]
Email:     [hidden email]
Website: http://home.golden.net/~lschafer/
____________________________________

 

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

From: [hidden email]

To: [hidden email]

Sent: Saturday, November 29, 2008 5:25 PM

Subject: Re: Deconvolution Algorithms Continued

 

I would like to thank everyone who replied to the Deconvolution Algorithm questions. Once I Get back to the lab on Monday we  will start comparing the suggested algorithms. Thanks Brian for your comments as well, the commercial product we are comparing against is the Autoquant module in the Nikon NIS elements software.

 

 I do have a question for all of you regarding calculating the point spread function.  For some of the open source algorithms we are using we have to input the PSF in the deconvolution algorithm.  For this we using a an open source software which calculates theoretical PSF after you input imaging parameters such as, magnification, NA etc. but this software does not have Laser Scanning Confocal microscopy as an imaging modality option, it only has wide field fluorescence. Is there a formula for theoretically  calculating PSF in which you can input  the Confocal pin hole as a parameter, or would it be easier to determine the PSF experimentally using fluorescent beads?

 

Best Regards,

 

Neeraj.

 

Neeraj V. Gohad, PhD

Postdoctoral Fellow,

Okeanos Research Group

Department of Biological Sciences

132 Long Hall,Clemson University

Clemson, SC-29634

864-656-3597

[hidden email]

 

[Filip Rooms]

> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Lutz Schaefer
> Sent: Saturday, November 29, 2008 5:55 PM
> To: [hidden email]
> Subject: Re: Deconvolution Algorithms Continued
>
> a confocal PSF (or generally, any incoherent PSF of an aplanatic microscope
> system) is the product of the excitation and emission distributions. The
> exitation PSF could be approximated by a widefield PSF and the emission PSF
> by a widefield PSF (using the emission wavelength) but convolved by the
> pinhole geometry. You need to be careful with sampling and scaling issues.
> Other than that its not a big deal. The approximation with widefield vs
> point-scanner had been okay in nearly all cases that came across my work.
> For the calculation of widefield PSF's I suggest the classic Sarah Gibson
> and Frad Lanni's paper "Experimental test of an analytical model of
> aberration in an oil immersion objective lens used in three dimensional
> light microscopy" JOSA V.9 N.1 1992 (which was to the best of my knowledge
> also implemented in XCOSM). But of course, you may also use the slightly
> more accurate vector wave model.

When I searched Google on "Geert Van Kempen", the first hit is a link to his
PhD: http://www.ns.tudelft.nl/live/pagina.jsp?id=d45bad79-71f7-4358-98d2-6bd78f432b3a&lang=en

In there, some explanation about image formation is given for both
widefield and
confocal microscopy (I used it as the main reference for a section in
my own thesis).

Kind regards,

Filip Rooms