Deconvolution advice
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, I'm looking for advice and information about deconvolution, especially from those with first-hand experience. Traditionally, one of the processing steps in structured illumination microscopy is deconvolution. For our SIM, we decided to use an open-source solution: https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution This seemed like a nice tradeoff between reinventing the wheel with our own deconvolution code, and subjecting ourselves to a 'black box' closed-source solution. However, we've recently tried out the Huygens deconvolution software, and the results seem quite promising, possibly an improvement over other methods we've tried. I like good images, but I don't like black boxes, and I like to understand my data processing. 1. Is the exact algorithm used in Huygens transparently documented anywhere? I spent a few hours searching today, but if it's out there, I missed it. 2. Is there a clear winner for deconvolution algorithms? What should I be using? 3. Are there other deconvolution software packages I should consider? Ideally I'm looking for software based on clearly-documented algorithms. Thanks for the help. -Andrew York NIH/NIBIB |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I have found the plugins by Bob Dougherty for ImageJ to be easy to use, and well thought-out. I don't know too much about the exact algorithms, but they might be worth a try. Joel Sheffield On Fri, Sep 28, 2012 at 4:33 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, I'm looking for advice and information about deconvolution, > especially from those with first-hand experience. > > Traditionally, one of the processing steps in structured illumination > microscopy is deconvolution. For our SIM, we decided to use an open-source > solution: > > https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution > > This seemed like a nice tradeoff between reinventing the wheel with our own > deconvolution code, and subjecting ourselves to a 'black box' closed-source > solution. However, we've recently tried out the Huygens deconvolution > software, and the results seem quite promising, possibly an improvement > over other methods we've tried. I like good images, but I don't like black > boxes, and I like to understand my data processing. > > 1. Is the exact algorithm used in Huygens transparently documented > anywhere? I spent a few hours searching today, but if it's out there, I > missed it. > > 2. Is there a clear winner for deconvolution algorithms? What should I be > using? > > 3. Are there other deconvolution software packages I should consider? > Ideally I'm looking for software based on clearly-documented algorithms. > > Thanks for the help. > > -Andrew York > NIH/NIBIB > -- Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] URL: http://astro.temple.edu/~jbs |
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In reply to this post by Andrew York
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Andrew, I don't have first-hand experience but I remember reading this two-part comparison of different deconvolution softwares, including commercial and open-source ones, in GIT Imaging & Microscopy by the Sage's group at EPFL (direct links to pdfs): http://bigwww.epfl.ch/publications/griffa1001.pdf http://bigwww.epfl.ch/publications/griffa1002.pdf Hope this helps, Christophe On Fri, Sep 28, 2012 at 10:33 PM, Andrew York < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, I'm looking for advice and information about deconvolution, > especially from those with first-hand experience. > > Traditionally, one of the processing steps in structured illumination > microscopy is deconvolution. For our SIM, we decided to use an open-source > solution: > > https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution > > This seemed like a nice tradeoff between reinventing the wheel with our own > deconvolution code, and subjecting ourselves to a 'black box' closed-source > solution. However, we've recently tried out the Huygens deconvolution > software, and the results seem quite promising, possibly an improvement > over other methods we've tried. I like good images, but I don't like black > boxes, and I like to understand my data processing. > > 1. Is the exact algorithm used in Huygens transparently documented > anywhere? I spent a few hours searching today, but if it's out there, I > missed it. > > 2. Is there a clear winner for deconvolution algorithms? What should I be > using? > > 3. Are there other deconvolution software packages I should consider? > Ideally I'm looking for software based on clearly-documented algorithms. > > Thanks for the help. > > -Andrew York > NIH/NIBIB > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Model, etal, Journal of Microscopy, Vol. 241, Pt 1 2011, pp. 94–100 another review of decon methods and influence of parameters. I believe the Huygens algorithm has been published. Just email them, [hidden email]. Regards, glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] On Sep 28, 2012, at 2:29 PM, Christophe Leterrier wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi Andrew, > > I don't have first-hand experience but I remember reading this two-part > comparison of different deconvolution softwares, including commercial and > open-source ones, in GIT Imaging & Microscopy by the Sage's group at EPFL > (direct links to pdfs): > > http://bigwww.epfl.ch/publications/griffa1001.pdf > http://bigwww.epfl.ch/publications/griffa1002.pdf > > Hope this helps, > > Christophe > > On Fri, Sep 28, 2012 at 10:33 PM, Andrew York < > [hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello, I'm looking for advice and information about deconvolution, >> especially from those with first-hand experience. >> >> Traditionally, one of the processing steps in structured illumination >> microscopy is deconvolution. For our SIM, we decided to use an open-source >> solution: >> >> https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution >> >> This seemed like a nice tradeoff between reinventing the wheel with our own >> deconvolution code, and subjecting ourselves to a 'black box' closed-source >> solution. However, we've recently tried out the Huygens deconvolution >> software, and the results seem quite promising, possibly an improvement >> over other methods we've tried. I like good images, but I don't like black >> boxes, and I like to understand my data processing. >> >> 1. Is the exact algorithm used in Huygens transparently documented >> anywhere? I spent a few hours searching today, but if it's out there, I >> missed it. >> >> 2. Is there a clear winner for deconvolution algorithms? What should I be >> using? >> >> 3. Are there other deconvolution software packages I should consider? >> Ideally I'm looking for software based on clearly-documented algorithms. >> >> Thanks for the help. >> >> -Andrew York >> NIH/NIBIB >> |
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In reply to this post by Andrew York
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Andrew- You might want to get in touch with Jim McNally. Last I heard he was at NIH-NCI. Some of his older papers on deconvoltion algorithms are below, but he can probably point you towards more recent information. http://www.ncbi.nlm.nih.gov/pubmed/10579932 http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 compared a 3D image processed by three different algorithms) Quoting Andrew York <[hidden email]>: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, I'm looking for advice and information about deconvolution, > especially from those with first-hand experience. > > Traditionally, one of the processing steps in structured illumination > microscopy is deconvolution. For our SIM, we decided to use an open-source > solution: > https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution > > This seemed like a nice tradeoff between reinventing the wheel with our own > deconvolution code, and subjecting ourselves to a 'black box' closed-source > solution. However, we've recently tried out the Huygens deconvolution > software, and the results seem quite promising, possibly an improvement > over other methods we've tried. I like good images, but I don't like black > boxes, and I like to understand my data processing. > > 1. Is the exact algorithm used in Huygens transparently documented > anywhere? I spent a few hours searching today, but if it's out there, I > missed it. > > 2. Is there a clear winner for deconvolution algorithms? What should I be > using? > > 3. Are there other deconvolution software packages I should consider? > Ideally I'm looking for software based on clearly-documented algorithms. > > Thanks for the help. > > -Andrew York > NIH/NIBIB > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** **commercial response** Hello Andrew, It's fully understandable that people want to know the scientific grounds when using Huygens. For the full list of articles I refer to http://www.svi.nl/HuygensReferences at which the relevant papers are at the bottom of the page and mostly written during the years 1996-1998. There are much more articles that ought to be included so your question shows that we must give more attention to this topic. With best wishes, Gitta Hamel **************************************** Gitta Hamel Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* software The Netherlands phone: ++ 31 35 6 42 16 26 ***************************************** ^SVI Customer support: mail us your questions [hidden email] <mailto:[hidden email]>or find answers online in our Huygens WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage> On 09/29/2012 04:00 PM, Gens, John Scott wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Andrew- > > You might want to get in touch with Jim McNally. Last I heard he was > at NIH-NCI. > > Some of his older papers on deconvoltion algorithms are below, but he > can probably point > you towards more recent information. > > http://www.ncbi.nlm.nih.gov/pubmed/10579932 > > http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 > compared a 3D image processed by three different algorithms) > > > Quoting Andrew York <[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello, I'm looking for advice and information about deconvolution, >> especially from those with first-hand experience. >> >> Traditionally, one of the processing steps in structured illumination >> microscopy is deconvolution. For our SIM, we decided to use an >> open-source >> solution: >> https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution >> >> >> This seemed like a nice tradeoff between reinventing the wheel with >> our own >> deconvolution code, and subjecting ourselves to a 'black box' >> closed-source >> solution. However, we've recently tried out the Huygens deconvolution >> software, and the results seem quite promising, possibly an improvement >> over other methods we've tried. I like good images, but I don't like >> black >> boxes, and I like to understand my data processing. >> >> 1. Is the exact algorithm used in Huygens transparently documented >> anywhere? I spent a few hours searching today, but if it's out there, I >> missed it. >> >> 2. Is there a clear winner for deconvolution algorithms? What should >> I be >> using? >> >> 3. Are there other deconvolution software packages I should consider? >> Ideally I'm looking for software based on clearly-documented algorithms. >> >> Thanks for the help. >> >> -Andrew York >> NIH/NIBIB >> > -- Managing Director Huygens SVI tel: +31 (0)35 642 16 26 fax: +31 (0)35 683 79 71 skype: gittahamel cell: +31(0)618 021272 Visiting address Laapersveld 63, 1213 VB Hilversum, The Netherlands |
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In reply to this post by Andrew York
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Andrew, Wang & Smith 2012 PLoS Comput Biology, http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1002671 used Richardson-Lucy built into MatLab. Had some nice results though did not deconvolve the competing methods, and botched the STED pixel size. George On 9/28/2012 4:33 PM, Andrew York wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, I'm looking for advice and information about deconvolution, > especially from those with first-hand experience. > > Traditionally, one of the processing steps in structured illumination > microscopy is deconvolution. For our SIM, we decided to use an open-source > solution: > https://sites.google.com/site/piotrwendykier/software/deconvolution/paralleliterativedeconvolution > > This seemed like a nice tradeoff between reinventing the wheel with our own > deconvolution code, and subjecting ourselves to a 'black box' closed-source > solution. However, we've recently tried out the Huygens deconvolution > software, and the results seem quite promising, possibly an improvement > over other methods we've tried. I like good images, but I don't like black > boxes, and I like to understand my data processing. > > 1. Is the exact algorithm used in Huygens transparently documented > anywhere? I spent a few hours searching today, but if it's out there, I > missed it. > > 2. Is there a clear winner for deconvolution algorithms? What should I be > using? > > 3. Are there other deconvolution software packages I should consider? > Ideally I'm looking for software based on clearly-documented algorithms. > > Thanks for the help. > > -Andrew York > NIH/NIBIB > > |
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In reply to this post by Gitta Hamel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, Deconvolution for SIM is a very different story than for other techniques. SIM by default uses an inverse filter in its reconstruction to recombine the shifted components of the fourier transform without enhancing the high frequency noise. Typical SIM software has a noise parameter for the wiener filter. If you set this filter low, you start to see a rippling pattern in the noise and eventually in the high signal regions. As far as I can tell, no one has tried to use more advanced (poisson noise driven) algorithms for this problem. If one assumes that Autoquant is using some variant of the algorithm shown in Tim Holmes' Handbook of Biological Confocal chapter, then this algorithm is not immediately applicable to the SIM problem. The algorithm update function involves dividing the original image by the convolved object guess and then convolving that ratio with the reflected psf and finally multiplying by the object guess. Given that the raw SIM image is actually a frequency modulated image in a particular direction, it is not clear how the original ratio would be generated. Richardson-Lucy has a similar problem. Would you deconvolve before reconstructing? This is the only circumstance under which the noise could be considered poisson. In that case, can the original PSF be used? I'm not entirely certain that the deconvolution would preserve the frequency modulation in the image. In addition, the original reconstruction algorithm would require a second step of deconvolution during the reconstruction step--not sure how the noise parameter should be chosen after initial deconvolution. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gitta Hamel Sent: Monday, October 01, 2012 8:42 AM To: [hidden email] Subject: Re: Deconvolution advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** **commercial response** Hello Andrew, It's fully understandable that people want to know the scientific grounds when using Huygens. For the full list of articles I refer to http://www.svi.nl/HuygensReferences at which the relevant papers are at the bottom of the page and mostly written during the years 1996-1998. There are much more articles that ought to be included so your question shows that we must give more attention to this topic. With best wishes, Gitta Hamel **************************************** Gitta Hamel Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* software The Netherlands phone: ++ 31 35 6 42 16 26 ***************************************** ^SVI Customer support: mail us your questions [hidden email] <mailto:[hidden email]>or find answers online in our Huygens WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage> On 09/29/2012 04:00 PM, Gens, John Scott wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Andrew- > > You might want to get in touch with Jim McNally. Last I heard he was > at NIH-NCI. > > Some of his older papers on deconvoltion algorithms are below, but he > can probably point you towards more recent information. > > http://www.ncbi.nlm.nih.gov/pubmed/10579932 > > http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 > compared a 3D image processed by three different algorithms) > > > Quoting Andrew York <[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello, I'm looking for advice and information about deconvolution, >> especially from those with first-hand experience. >> >> Traditionally, one of the processing steps in structured illumination >> microscopy is deconvolution. For our SIM, we decided to use an >> open-source >> solution: >> https://sites.google.com/site/piotrwendykier/software/deconvolution/p >> aralleliterativedeconvolution >> >> >> This seemed like a nice tradeoff between reinventing the wheel with >> our own deconvolution code, and subjecting ourselves to a 'black box' >> closed-source >> solution. However, we've recently tried out the Huygens deconvolution >> software, and the results seem quite promising, possibly an >> improvement over other methods we've tried. I like good images, but I >> don't like black boxes, and I like to understand my data processing. >> >> 1. Is the exact algorithm used in Huygens transparently documented >> anywhere? I spent a few hours searching today, but if it's out there, >> I missed it. >> >> 2. Is there a clear winner for deconvolution algorithms? What should >> I be using? >> >> 3. Are there other deconvolution software packages I should consider? >> Ideally I'm looking for software based on clearly-documented algorithms. >> >> Thanks for the help. >> >> -Andrew York >> NIH/NIBIB >> > -- Managing Director Huygens SVI tel: +31 (0)35 642 16 26 fax: +31 (0)35 683 79 71 skype: gittahamel cell: +31(0)618 021272 Visiting address Laapersveld 63, 1213 VB Hilversum, The Netherlands |
 
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Kevin Ryan |
Re: Deconvolution advice - Commercial response
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** **COMMERCIAL RESPONSE, MEDIA CYBERNETICS - AUTOQUANT ** Jay is correct that imaging modes such as SIM will not have PSFs matching ordinary microscopic modes. For deconvolving novel imaging methods such as SIM, the first and best approach is probably to use a fixed, measured PSF: * Image a point object under the same conditions as your desired sample (and that includes your _embedding media_, as the refractive index of your media is a critical element of image formation!), then use that as a model for the system PSF. That should incorporate all elements of the imaging system in the transform between the point object and the observed PSF. The better the PSF image, the better final reconstruction will be. As an example, AutoQuant has been used in this fashion for computed holographic microscopy (Rosen and Brooker 2008, "Non-scanning motionless fluorescence three-dimensional holographic microscopy", http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.153.3691&rep=rep1&type=pdf), with quite good results. The PSF for that system is notably _not_ a standard widefield or confocal blur. * Blind deconvolution, where the PSF is optimized along with the data, _can_ be done with novel microscopic imaging modes by starting with a measured PSF and disabling PSF constraints (which are the constraints appropriate for widefield/confocal data - radial symmetry, energy conservation, etc). That would, however, be an experimental approach, and should be checked for ground truth against known specimens to see if it is appropriate for your data. Examine both the resulting deconvolution for known structures (did it remove blur, improve resolution?), and the resulting PSF for comparison to using SIM on a point object (does the final PSF have the general form of a measured point object?). This may provide a reconstruction improvement over a measured PSF by incorporating information from the observed sample. Kevin Ryan Media Cybernetics, Inc. -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Unruh, Jay Sent: Tuesday, October 02, 2012 10:18 AM To: [hidden email] Subject: Re: Deconvolution advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi all, Deconvolution for SIM is a very different story than for other techniques. SIM by default uses an inverse filter in its reconstruction to recombine the shifted components of the fourier transform without enhancing the high frequency noise. Typical SIM software has a noise parameter for the wiener filter. If you set this filter low, you start to see a rippling pattern in the noise and eventually in the high signal regions. As far as I can tell, no one has tried to use more advanced (poisson noise driven) algorithms for this problem. If one assumes that Autoquant is using some variant of the algorithm shown in Tim Holmes' Handbook of Biological Confocal chapter, then this algorithm is not immediately applicable to the SIM problem. The algorithm update function involves dividing the original image by the convolved object guess and then convolving that ratio with the reflected psf and finally multiplying by the object guess. Given that the raw SIM image is actually a frequency modulated image in a particular direction, it is not clear how the original ratio would be generated. Richardson-Lucy has a similar problem. Would you deconvolve before reconstructing? This is the only circumstance under which the noise could be considered poisson. In that case, can the original PSF be used? I'm not entirely certain that the deconvolution would preserve the frequency modulation in the image. In addition, the original reconstruction algorithm would require a second step of deconvolution during the reconstruction step--not sure how the noise parameter should be chosen after initial deconvolution. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gitta Hamel Sent: Monday, October 01, 2012 8:42 AM To: [hidden email] Subject: Re: Deconvolution advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** **commercial response** Hello Andrew, It's fully understandable that people want to know the scientific grounds when using Huygens. For the full list of articles I refer to http://www.svi.nl/HuygensReferences at which the relevant papers are at the bottom of the page and mostly written during the years 1996-1998. There are much more articles that ought to be included so your question shows that we must give more attention to this topic. With best wishes, Gitta Hamel **************************************** Gitta Hamel Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* software The Netherlands phone: ++ 31 35 6 42 16 26 ***************************************** ^SVI Customer support: mail us your questions [hidden email] <mailto:[hidden email]>or find answers online in our Huygens WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage> On 09/29/2012 04:00 PM, Gens, John Scott wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Andrew- > > You might want to get in touch with Jim McNally. Last I heard he was > at NIH-NCI. > > Some of his older papers on deconvoltion algorithms are below, but he > can probably point you towards more recent information. > > http://www.ncbi.nlm.nih.gov/pubmed/10579932 > > http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 > compared a 3D image processed by three different algorithms) > > > Quoting Andrew York <[hidden email]>: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello, I'm looking for advice and information about deconvolution, >> especially from those with first-hand experience. >> >> Traditionally, one of the processing steps in structured illumination >> microscopy is deconvolution. For our SIM, we decided to use an >> open-source >> solution: >> https://sites.google.com/site/piotrwendykier/software/deconvolution/p >> aralleliterativedeconvolution >> >> >> This seemed like a nice tradeoff between reinventing the wheel with >> our own deconvolution code, and subjecting ourselves to a 'black box' >> closed-source >> solution. However, we've recently tried out the Huygens deconvolution >> software, and the results seem quite promising, possibly an >> improvement over other methods we've tried. I like good images, but I >> don't like black boxes, and I like to understand my data processing. >> >> 1. Is the exact algorithm used in Huygens transparently documented >> anywhere? I spent a few hours searching today, but if it's out there, >> I missed it. >> >> 2. Is there a clear winner for deconvolution algorithms? What should >> I be using? >> >> 3. Are there other deconvolution software packages I should consider? >> Ideally I'm looking for software based on clearly-documented algorithms. >> >> Thanks for the help. >> >> -Andrew York >> NIH/NIBIB >> > -- Managing Director Huygens SVI tel: +31 (0)35 642 16 26 fax: +31 (0)35 683 79 71 skype: gittahamel cell: +31(0)618 021272 Visiting address Laapersveld 63, 1213 VB Hilversum, The Netherlands ###################################################################################### CONFIDENTIALITY NOTICE: This email transmission and its attachments contain confidential and proprietary information of Princeton Instruments, Acton Research, Media Cybernetics and their affiliates and is intended for the exclusive and confidential use of the intended recipient. Any use, dissemination, printing, or copying of this transmission and its attachment(s) is strictly prohibited. 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In reply to this post by Unruh, Jay
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello all, Jay is correct, a regularized inverse filter of the separated (shifted) orders can be used followed by magnitude (and/or linear) reconstruction. To the best of my knowledge, this principle is implemented in the Zeiss ZEN and AxioVision software. It is also possible to use a Gauss likelihood iterative algorithm, but to no advantage as the extracted orders cannot be constrained to positivity. A realization of Poisson likelihood will, as Jay says proof difficult, owing to its inherent nonlinearity. Best Regards Lutz __________________________________ L u t z S c h a e f e r Sen. Scientist Mathematical modeling / Image processing Advanced Imaging Methodology Consultation 16-715 Doon Village Rd. Kitchener, ON, N2P 2A2, Canada Phone/Fax: +1 519 894 8870 Email: [hidden email] ___________________________________ -------------------------------------------------- From: "Unruh, Jay" <[hidden email]> Sent: Tuesday, October 02, 2012 10:17 To: <[hidden email]> Subject: Re: Deconvolution advice > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi all, > > Deconvolution for SIM is a very different story than for other techniques. > SIM by default uses an inverse filter in its reconstruction to recombine > the shifted components of the fourier transform without enhancing the high > frequency noise. Typical SIM software has a noise parameter for the > wiener filter. If you set this filter low, you start to see a rippling > pattern in the noise and eventually in the high signal regions. As far as > I can tell, no one has tried to use more advanced (poisson noise driven) > algorithms for this problem. > > If one assumes that Autoquant is using some variant of the algorithm shown > in Tim Holmes' Handbook of Biological Confocal chapter, then this > algorithm is not immediately applicable to the SIM problem. The algorithm > update function involves dividing the original image by the convolved > object guess and then convolving that ratio with the reflected psf and > finally multiplying by the object guess. Given that the raw SIM image is > actually a frequency modulated image in a particular direction, it is not > clear how the original ratio would be generated. Richardson-Lucy has a > similar problem. Would you deconvolve before reconstructing? This is the > only circumstance under which the noise could be considered poisson. In > that case, can the original PSF be used? I'm not entirely certain that > the deconvolution would preserve the frequency modulation in the image. > In addition, the original reconstruction algorithm would require a second > step of deconvolution during the reconstruction step--not sure how the > noise parameter should be chosen after initial deconvolution. > > Jay > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Gitta Hamel > Sent: Monday, October 01, 2012 8:42 AM > To: [hidden email] > Subject: Re: Deconvolution advice > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > **commercial response** > > > Hello Andrew, > > It's fully understandable that people want to know the scientific grounds > when using Huygens. > > For the full list of articles I refer to > http://www.svi.nl/HuygensReferences at which the relevant papers are at > the bottom of the page and mostly written during the years 1996-1998. > There are much more articles that ought to be included so your question > shows that we must give more attention to this topic. > > With best wishes, > > Gitta Hamel > > **************************************** > Gitta Hamel > Managing Director Scientific Volume Imaging bv Developers of the *HUYGENS* > software The Netherlands > phone: ++ 31 35 6 42 16 26 > ***************************************** > > > ^SVI Customer support: mail us your questions [hidden email] > <mailto:[hidden email]>or find answers online in our Huygens > WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage> > > > > On 09/29/2012 04:00 PM, Gens, John Scott wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Andrew- >> >> You might want to get in touch with Jim McNally. Last I heard he was >> at NIH-NCI. >> >> Some of his older papers on deconvoltion algorithms are below, but he >> can probably point you towards more recent information. >> >> http://www.ncbi.nlm.nih.gov/pubmed/10579932 >> >> http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 >> compared a 3D image processed by three different algorithms) >> >> >> Quoting Andrew York <[hidden email]>: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hello, I'm looking for advice and information about deconvolution, >>> especially from those with first-hand experience. >>> >>> Traditionally, one of the processing steps in structured illumination >>> microscopy is deconvolution. For our SIM, we decided to use an >>> open-source >>> solution: >>> https://sites.google.com/site/piotrwendykier/software/deconvolution/p >>> aralleliterativedeconvolution >>> >>> >>> This seemed like a nice tradeoff between reinventing the wheel with >>> our own deconvolution code, and subjecting ourselves to a 'black box' >>> closed-source >>> solution. However, we've recently tried out the Huygens deconvolution >>> software, and the results seem quite promising, possibly an >>> improvement over other methods we've tried. I like good images, but I >>> don't like black boxes, and I like to understand my data processing. >>> >>> 1. Is the exact algorithm used in Huygens transparently documented >>> anywhere? I spent a few hours searching today, but if it's out there, >>> I missed it. >>> >>> 2. Is there a clear winner for deconvolution algorithms? What should >>> I be using? >>> >>> 3. Are there other deconvolution software packages I should consider? >>> Ideally I'm looking for software based on clearly-documented algorithms. >>> >>> Thanks for the help. >>> >>> -Andrew York >>> NIH/NIBIB >>> >> > > -- > Managing Director > > Huygens SVI > > tel: +31 (0)35 642 16 26 > > fax: +31 (0)35 683 79 71 > > skype: gittahamel > > cell: +31(0)618 021272 > > Visiting address > > Laapersveld 63, > 1213 VB Hilversum, > The Netherlands |
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George McNamara |
Re: Deconvolution advice ... MSIM is "a lot like a point-scanning confocal with twice the resolution and higher signal-to-noise"
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Jay and Lutz, I believe Andrew is not going to deconvolve the stripes, instead he is going to deconvolve the points - see video 1 at http://code.google.com/p/msim/ the ... in the subject line is from: That's what 'msim' does. Specifically, we built a new kind of fluorescence microscope, which we call a 'multifocal structured illumination microscope' (MSIM). Distinguishing features: * 3D superresolution (145 nm transverse, 400 nm axial resolution) * Uses standard fluorescent probes (like GFP), currently configured for 488 and 561 nm illumination * Works in thick samples (>50 microns). Previous SIM scopes couldn't do this. * Reasonable speed (1 2D slice per second) * Live-cell compatible (tens or hundreds of 3D volumes) * Low photobleaching (similar to a spinning disk confocal) * 50x50 micron field of view Functionally, it's a lot like a point-scanning confocal with twice the resolution and higher signal-to-noise, at the cost of digital post-processing. His Nature Methods paper http://www.nature.com/nmeth/journal/v9/n7/full/nmeth.2025.html mentions that the 3D superresolution values he measured, 145 nm XY, 300 nm Z, was with a standard objective lens - not hand picked: "The apparent full-width at half maximum (FWHM) intensity of microtubules in MSIM images was 145 nm, a twofold improvement compared to wide-field imaging (Fig. 1d). Similar experiments on 110-nm beads confirmed this result (MSIM FWHM of 146 nm ± 15 nm versus wide-field FWHM of 284 nm ± 32 nm (± s.d.), n = 80 beads, Supplementary Fig. 7). The acquisition time for the 48 ?m × 49 ?m field (Fig. 1a) was ~1 s, more than a 6,500-fold improvement over ISM, assuming the same 222-Hz raw frame rate." and, Like existing 3D SIM16, MSIM provides resolution doubling by increasing the highest spatial frequencies encoded in the raw data, followed by deconvolution. However, the absolute resolutions we report are slightly lower than existing 3D SIM, which we attribute to two factors. First, achieving the highest possible resolution relies on obtaining diffraction-limited wide-field performance before (M)SIM is applied. Although we attempted this, the wide-field point spread functions (PSFs) we measured (Supplementary Fig. 7) had FWHM (~280 nm) larger than theoretical predictions. It is customary in SIM to screen many objectives and use only the one with highest resolution. We did not do this. Furthermore, we suspect that aberration caused by warping of the dichroic in our microscope was the major cause of departure from ideal performance, as we noticed that small adjustments of the mounting screws that held the dichroic drastically altered the PSF. A thicker, stiffer dichroic may mitigate this issue. Second, the excitation patterns in previous SIM implementations concentrate energy at the very highest lateral spatial frequencies in the objective pass-band. In contrast, the multifocal excitation patterns we used contain all spatial frequencies permitted by the objective. We might thus expect line-excitation--based SIM to provide a greater SNR at high spatial frequencies than MSIM, enabling higher resolution. We note the point-like nature of our excitation pattern offers a route to even higher resolution, perhaps by exploiting the nonlinearities provided by reversible, switchable fluorescent probes29. Compared to previous SIM implementations, our system is simpler to build. We add only a DMD, a telescope and a fast camera to a conventional wide-field microscope. Our illumination path has no moving parts and is insensitive to polarization. Finally, we do not modify the illumination path for multicolor operation, unlike existing SIM implementations30. Upshot: MSIM is likely to get even better! George On 10/2/2012 12:40 PM, Lutz Schaefer wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello all, > > Jay is correct, a regularized inverse filter of the separated > (shifted) orders can be used followed by magnitude (and/or linear) > reconstruction. To the best of my knowledge, this principle is > implemented in the Zeiss ZEN and AxioVision software. It is also > possible to use a Gauss likelihood iterative algorithm, but to no > advantage as the extracted orders cannot be constrained to positivity. > A realization of Poisson likelihood will, as Jay says proof difficult, > owing to its inherent nonlinearity. > > Best Regards > Lutz > > __________________________________ > L u t z S c h a e f e r > Sen. Scientist > Mathematical modeling / Image processing > Advanced Imaging Methodology Consultation > 16-715 Doon Village Rd. > Kitchener, ON, N2P 2A2, Canada > Phone/Fax: +1 519 894 8870 > Email: [hidden email] > ___________________________________ > > -------------------------------------------------- > From: "Unruh, Jay" <[hidden email]> > Sent: Tuesday, October 02, 2012 10:17 > To: <[hidden email]> > Subject: Re: Deconvolution advice > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi all, >> >> Deconvolution for SIM is a very different story than for other >> techniques. SIM by default uses an inverse filter in its >> reconstruction to recombine the shifted components of the fourier >> transform without enhancing the high frequency noise. Typical SIM >> software has a noise parameter for the wiener filter. If you set >> this filter low, you start to see a rippling pattern in the noise and >> eventually in the high signal regions. As far as I can tell, no one >> has tried to use more advanced (poisson noise driven) algorithms for >> this problem. >> >> If one assumes that Autoquant is using some variant of the algorithm >> shown in Tim Holmes' Handbook of Biological Confocal chapter, then >> this algorithm is not immediately applicable to the SIM problem. The >> algorithm update function involves dividing the original image by the >> convolved object guess and then convolving that ratio with the >> reflected psf and finally multiplying by the object guess. Given >> that the raw SIM image is actually a frequency modulated image in a >> particular direction, it is not clear how the original ratio would be >> generated. Richardson-Lucy has a similar problem. Would you >> deconvolve before reconstructing? This is the only circumstance >> under which the noise could be considered poisson. In that case, can >> the original PSF be used? I'm not entirely certain that the >> deconvolution would preserve the frequency modulation in the image. >> In addition, the original reconstruction algorithm would require a >> second step of deconvolution during the reconstruction step--not sure >> how the noise parameter should be chosen after initial deconvolution. >> >> Jay >> >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On Behalf Of Gitta Hamel >> Sent: Monday, October 01, 2012 8:42 AM >> To: [hidden email] >> Subject: Re: Deconvolution advice >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> **commercial response** >> >> >> Hello Andrew, >> >> It's fully understandable that people want to know the scientific >> grounds when using Huygens. >> >> For the full list of articles I refer to >> http://www.svi.nl/HuygensReferences at which the relevant papers are >> at the bottom of the page and mostly written during the years 1996-1998. >> There are much more articles that ought to be included so your >> question shows that we must give more attention to this topic. >> >> With best wishes, >> >> Gitta Hamel >> >> **************************************** >> Gitta Hamel >> Managing Director Scientific Volume Imaging bv Developers of the >> *HUYGENS* software The Netherlands >> phone: ++ 31 35 6 42 16 26 >> ***************************************** >> >> >> ^SVI Customer support: mail us your questions [hidden email] >> <mailto:[hidden email]>or find answers online in our Huygens >> WIKI:www.svi.nl/FrontPage <http://%20www.svi.nl/FrontPage> >> >> >> >> On 09/29/2012 04:00 PM, Gens, John Scott wrote: >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Andrew- >>> >>> You might want to get in touch with Jim McNally. Last I heard he was >>> at NIH-NCI. >>> >>> Some of his older papers on deconvoltion algorithms are below, but he >>> can probably point you towards more recent information. >>> >>> http://www.ncbi.nlm.nih.gov/pubmed/10579932 >>> >>> http://www.ncbi.nlm.nih.gov/pubmed/11541650 ( in particular, fig.2 >>> compared a 3D image processed by three different algorithms) >>> >>> >>> Quoting Andrew York <[hidden email]>: >>> >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> ***** >>>> >>>> Hello, I'm looking for advice and information about deconvolution, >>>> especially from those with first-hand experience. >>>> >>>> Traditionally, one of the processing steps in structured illumination >>>> microscopy is deconvolution. For our SIM, we decided to use an >>>> open-source >>>> solution: >>>> https://sites.google.com/site/piotrwendykier/software/deconvolution/p >>>> aralleliterativedeconvolution >>>> >>>> >>>> This seemed like a nice tradeoff between reinventing the wheel with >>>> our own deconvolution code, and subjecting ourselves to a 'black box' >>>> closed-source >>>> solution. However, we've recently tried out the Huygens deconvolution >>>> software, and the results seem quite promising, possibly an >>>> improvement over other methods we've tried. I like good images, but I >>>> don't like black boxes, and I like to understand my data processing. >>>> >>>> 1. Is the exact algorithm used in Huygens transparently documented >>>> anywhere? I spent a few hours searching today, but if it's out there, >>>> I missed it. >>>> >>>> 2. Is there a clear winner for deconvolution algorithms? What should >>>> I be using? >>>> >>>> 3. Are there other deconvolution software packages I should consider? >>>> Ideally I'm looking for software based on clearly-documented >>>> algorithms. >>>> >>>> Thanks for the help. >>>> >>>> -Andrew York >>>> NIH/NIBIB >>>> >>> >> >> -- >> Managing Director >> >> Huygens SVI >> >> tel: +31 (0)35 642 16 26 >> >> fax: +31 (0)35 683 79 71 >> >> skype: gittahamel >> >> cell: +31(0)618 021272 >> >> Visiting address >> >> Laapersveld 63, >> 1213 VB Hilversum, >> The Netherlands > |
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In reply to this post by Lutz Schaefer
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Andrew As previously discussed there are a multitude of issues to consider when deconvolving the images from your customized microscope such as choosing an algorithm appropriate for the noise model. While investigating that you would also want to understand any noise related user-adjustable parameters the algorithms may have. After you've worked through the noise handling of the algorithm you would want to look very carefully at the PSF generation. I was with AutoQuant/MediaCy for many years and worked on (among other things) theoretical psf and aberration models, so I have some thoughts on these issues. As others have mentioned for a novel modality, if no software is available for calculating a theoretical PSF, you would have to use a measured PSF. From being involved in deconvolution in the past and attending the same workshops as SVI I am moderately familiar with Huygens. One possibility is that the differences you are seeing between the Huygens implementation and the Parallel Iterative open source implementation could be partially due to PSF processing. You should ask SVI more about this part. As you've probably experienced the measured PSF needs to be preconditioned. At the very least it should be centred and made the same size as the image. It is sometimes denoised and made (at least laterally-radially) symmetric. There are many variations of psf preprocessing routines. Some involve preconditioning a single bead. Some average multiple beads. Another alternative is to solve for the PSF using a "reverse deconvolution" on an object of known size and shape (usually a field of beads which in this case do not need to be sub resolution). Huygens has the option of solving for the PSF through a reverse deconvolution scheme they call "distilling". So it could be that the PSF pre-processing and/or distilling (if you are using it) in the Huygens software is making a difference. I don't know for sure but it would be something to investigate. You should see if Huygens allows you to export the preprocessed/distilled PSF and if so take a look at it. Brian |
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