General question: Software vs. hardware
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi. There are software solutions that are able to create image data from wide-field and even microscope systems with seemingly similar quality to that obtained from confocal systems. The comparison of acquisition vs. software is essentially a comparison of image acquisition vs. image processing. While a software solution is way cheaper than a hardware solution if it is able to produce image data with equal quality why would anyone choose to invest to a confocal anymore? I’m looking forward to a vidid discussion! >m |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Mike, Are you talking about deconvolving widefield fluorescence stacks? Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Mika Ruonala Sent: Sunday, March 17, 2019 10:44 AM To: [hidden email] Subject: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi. There are software solutions that are able to create image data from wide-field and even microscope systems with seemingly similar quality to that obtained from confocal systems. The comparison of acquisition vs. software is essentially a comparison of image acquisition vs. image processing. While a software solution is way cheaper than a hardware solution if it is able to produce image data with equal quality why would anyone choose to invest to a confocal anymore? I’m looking forward to a vidid discussion! >m |
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In reply to this post by ICIT
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Essentially, this is the question. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** This is a very old debate. I prefer confocal - fewer artifacts. Mike M -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Mika Ruonala Sent: Sunday, March 17, 2019 11:17 AM To: [hidden email] Subject: Re: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Essentially, this is the question. |
 
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George McNamara |
Re: General question: Software vs. hardware
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In reply to this post by ICIT
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Mika, White et al 1987 ( http://jcb.rupress.org/content/105/1/41.long ) made a compelling case for point scanning confocal microscopes: collect just the in focus light with instant gratification. The case has not changed, the hardware (especially data deluge side) has gotten a lot better. I note that both widefield detectors and PMT/APD/hybrid detectors/others have gotten a lot better in the 32 years from 1987! As have the optics and automation. Paul Goodwin 2014 ( https://www.ncbi.nlm.nih.gov/pubmed/24974028 ) made a nice case for quantitative deconvolution microscopy (and on very high quality specimens, ~10% improvement in resolution compared to simple widefield), but historically slow. Now, with 'instant gratification' spatial deconvolution, thanks to the GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 ... not including the deconvolution software module price), widefield, spinning disk (and slightly exotic variants like iSIM, DMD based, etc, see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited about the price point of recent fiber lasers, which could become much better price if achieve 'economy of scale'), and of course, point scanning confocal microscopes. Spatial deconvolution (especially if someone $uccessfully implements joint spatial deconvolution and spectral unmixing, multiple cameras - for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... really single molecule counting (and DNA-PAINT eliminates the classic issue of PALM/STORM/FPALM of not counting every molecule). Sure, DNA-PAINT (like STORM etc) have the issue of a whole lotta images acquired. Data deluge: who cares? Jerome & Price's 10th commandment of confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". More significantly, DNA-PAINT and related methods (single molecule RNA FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable multiplex -- with single molecule counting -- to whatever plex is needed to answer the 'biological question(s)' being posed. All that said, the installed base of research grade point scanning confocal microscopes is large (5000+) and efficient at acquiring high quality images, to the point that user's sample preparation (and avoidance of purchasing stuff from 'Santa Crap' and similar companies) is much more limiting than the microscopes. George p.s. a couple of references not included in above: W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy second edition https://link.springer.com/book/10.1007%2F978-3-319-97454-5 Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, https://www.nature.com/articles/s41596-018-0117-3 DNA-PAINT acronym soup review ... Nieves 2018 Genes, https://www.mdpi.com/2073-4425/9/12/621 Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy with $1550 CMOS cameras) ... https://www.nature.com/articles/s41598-018-19981-z Moffitt et al 2018 ... http://science.sciencemag.org/content/362/6416/eaau5324.long and commentary http://science.sciencemag.org/content/362/6416/749 *** Some resolution numbers: 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength / NA (I routinely drop the 0.61 from 0.61) 0.6 * 500 / 1.4 = 214 nm widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if pixel size matched or interpolate optimally). point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles - Confocal Laser Scanning Microscopy" (see fig 10) on confocal resolution wrt 1 and smaller pinhole size, source of the values below, 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput (which doesn't matter if target is photostable). 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and FastAiryScan). 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. Most modern point scanning confocal microscopes have a 405 nm laser, so if using BV421 (and ignoring potential photobleaching for a moment), 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. or in reflection mode, i.e. nanodiamond or nanogold, 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no photobleaching, so infinite number of photons (though also no blinking, so not usually eligible for precision localization) ... 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm and not going completely exotic with NA (i.e. 1.65), if perfectly refractive index match with a fairly conventional 1.49 NA lens, and inreflectance: 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to 101 nm. DNA-PAINT makes resolution irrelevant, if you use it (and don't run out of disk space or time or money), since precision localization is resolution divided by square root of number of photons, ex. 250 nm XY resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of photons per target further, but why bother? *** point scanning confocal microscopes are also great platforms for F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, https://www.ncbi.nlm.nih.gov/pubmed/18387308 *** p.p.s. Disclosures I am ... 1. currently hosting a Leica THUNDER Imager tour event (ends Monday 3/18/2019 afternoon) ... see pdf download page, THUNDER Technology Note THUNDER Imagers: How Do They Really Work? https://www.leica-microsystems.com/science-lab/thunder-technology-note 2. hosting Nikon confocal demos in May 2019. 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this summer. 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: https://www.ncbi.nlm.nih.gov/pubmed/28261321 On 3/17/2019 10:43 AM, Mika Ruonala wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi. > There are software solutions that are able to create image data from wide-field and even microscope systems with seemingly similar quality to that obtained from confocal systems. > > The comparison of acquisition vs. software is essentially a comparison of image acquisition vs. image processing. While a software solution is way cheaper than a hardware solution if it is able to produce image data with equal quality why would anyone choose to invest to a confocal anymore? > > I’m looking forward to a vidid discussion! > >> m |
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Sylvie Le Guyader |
Re: General question: Software vs. hardware
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In reply to this post by mmodel
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Or about the Leica Thunder? It would be good to know what they do to judge the risk for artefacts. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Hälsovägen 7C, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL Sent: 17 March 2019 16:04 To: [hidden email] Subject: Re: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Mike, Are you talking about deconvolving widefield fluorescence stacks? Mike Model -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Mika Ruonala Sent: Sunday, March 17, 2019 10:44 AM To: [hidden email] Subject: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi. There are software solutions that are able to create image data from wide-field and even microscope systems with seemingly similar quality to that obtained from confocal systems. The comparison of acquisition vs. software is essentially a comparison of image acquisition vs. image processing. While a software solution is way cheaper than a hardware solution if it is able to produce image data with equal quality why would anyone choose to invest to a confocal anymore? I’m looking forward to a vidid discussion! >m När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>. |
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In reply to this post by George McNamara
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I'll point out, that you can, of course, deconvolve confocal images too. So, while you can indeed get near confocal quality with well-acquired wf data after deconvolution, you can also get near SR quality when deconvolving a well-acquired stack from a confocal. And then you can also deconvolve a SR stack and get... well you get the idea! It's like an arms race. I have the Hyvolution and had access for a couple of weeks to the Lighting, and now confocal images look blurry to me. Avi -- Avi Jacob, Ph.D. Kanbar Light Microscopy Unit The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 5290002, Israel On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Mika, > > White et al 1987 ( http://jcb.rupress.org/content/105/1/41.long ) made a > compelling case for point scanning confocal microscopes: collect just > the in focus light with instant gratification. The case has not changed, > the hardware (especially data deluge side) has gotten a lot better. I > note that both widefield detectors and PMT/APD/hybrid detectors/others > have gotten a lot better in the 32 years from 1987! As have the optics > and automation. > > Paul Goodwin 2014 ( https://www.ncbi.nlm.nih.gov/pubmed/24974028 ) made > a nice case for quantitative deconvolution microscopy (and on very high > quality specimens, ~10% improvement in resolution compared to simple > widefield), but historically slow. > > Now, with 'instant gratification' spatial deconvolution, thanks to the > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > ... not including the deconvolution software module price), widefield, > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > about the price point of recent fiber lasers, which could become much > better price if achieve 'economy of scale'), and of course, point > scanning confocal microscopes. > > Spatial deconvolution (especially if someone $uccessfully implements > joint spatial deconvolution and spectral unmixing, multiple cameras - > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > really single molecule counting (and DNA-PAINT eliminates the classic > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > More significantly, DNA-PAINT and related methods (single molecule RNA > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > multiplex -- with single molecule counting -- to whatever plex is needed > to answer the 'biological question(s)' being posed. > > All that said, the installed base of research grade point scanning > confocal microscopes is large (5000+) and efficient at acquiring high > quality images, to the point that user's sample preparation (and > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > is much more limiting than the microscopes. > > George > > p.s. a couple of references not included in above: > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy > second edition https://link.springer.com/book/10.1007%2F978-3-319-97454-5 > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > https://www.nature.com/articles/s41596-018-0117-3 > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > https://www.mdpi.com/2073-4425/9/12/621 > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > with $1550 CMOS cameras) ... > https://www.nature.com/articles/s41598-018-19981-z > > Moffitt et al 2018 ... > http://science.sciencemag.org/content/362/6416/eaau5324.long and > commentary http://science.sciencemag.org/content/362/6416/749 > > *** > > Some resolution numbers: > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength > / NA (I routinely drop the 0.61 from 0.61) > > 0.6 * 500 / 1.4 = 214 nm > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > pixel size matched or interpolate optimally). > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > resolution wrt 1 and smaller pinhole size, source of the values below, > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput > (which doesn't matter if target is photostable). > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > FastAiryScan). > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > Most modern point scanning confocal microscopes have a 405 nm laser, so > if using BV421 (and ignoring potential photobleaching for a moment), > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > or in reflection mode, i.e. nanodiamond or nanogold, > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > photobleaching, so infinite number of photons (though also no blinking, > so not usually eligible for precision localization) ... > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > and not going completely exotic with NA (i.e. 1.65), if perfectly > refractive index match with a fairly conventional 1.49 NA lens, and > inreflectance: > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to > 101 nm. > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out > of disk space or time or money), since precision localization is > resolution divided by square root of number of photons, ex. 250 nm XY > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > photons per target further, but why bother? > > *** > > point scanning confocal microscopes are also great platforms for > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > https://www.ncbi.nlm.nih.gov/pubmed/18387308 > > *** > > p.p.s. Disclosures I am ... > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > 3/18/2019 afternoon) ... see pdf download page, > > THUNDER Technology Note > THUNDER Imagers: How Do They Really Work? > > https://www.leica-microsystems.com/science-lab/thunder-technology-note > > 2. hosting Nikon confocal demos in May 2019. > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > summer. > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > https://www.ncbi.nlm.nih.gov/pubmed/28261321 > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Hi. > > There are software solutions that are able to create image data from > wide-field and even microscope systems with seemingly similar quality to > that obtained from confocal systems. > > > > The comparison of acquisition vs. software is essentially a comparison > of image acquisition vs. image processing. While a software solution is way > cheaper than a hardware solution if it is able to produce image data with > equal quality why would anyone choose to invest to a confocal anymore? > > > > I’m looking forward to a vidid discussion! > > > >> m > |
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Glyn Nelson |
Re: General question: Software vs. hardware
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In reply to this post by ICIT
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** To throw my ha'penn'eth's worth in, I am on the side of confocal for a lot of the same reasons: no artefacts in the first place, plus deconvolution can be applied to confocal images for a minor improvement (and I believe is recommended in Pawley's excellent book). All of the things that would make a widefield system comparable (multiple cameras, fast GPUs, pixel shift free filtercubes etc) just bring the price of the widefield close to a confocal anyway. Admittedly the running costs are cheaper though. So to summarise- confocal + deconvolution is my preference. Glyn |
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Talley Lambert |
Re: General question: Software vs. hardware
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In reply to this post by ICIT
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** As Mike said, this is indeed a very old debate, but there are well-characterized, objective costs and benefits to each approach (scientific, not just monetary). Deconvolution is not just "computational confocal at the expense of artifacts"... it's a false comparison. By rejecting out-of-focus fluorescence, confocal microscopes reduce the *shot noise* contributed to the image by background. Deconvolution, by contrast attempts "reassign" that out of focus information (provided you have a very accurate representation of the actual PSF in your sample...), but there will come a point with thicker samples at which the shot noise contributed by out-of-focus fluorescence overwhelms the SNR in the image, and deconvolution will fail (figure 4 in the first paper below). However, for thin samples with minimal out-of-focus fluorescence, the increased collection efficiency and minimized illumination/detector noise of widefield+decon has benefits for detection of weak signals (figure 2 in the paper below). This tradeoff was well-characterized by Swedlow and Murray https://www.ncbi.nlm.nih.gov/pubmed/11830634 ...and followed up with a treatment on the photon-efficiency of different optical sectioning techniques: https://www.ncbi.nlm.nih.gov/pubmed/18045334 As usual, there is no one technique that is universally "better" or preferable. It will depend on the samples you are imaging and the relative levels of in-focus and out-of-focus information. |
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Tim Feinstein |
Re: General question: Software vs. hardware
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In reply to this post by Avi Jacob
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Avi, I really agree with your point. I feel that people to deconvolve any time spatial information is critical, whether they're using widefield, CLSM, spinning disc, or light sheet. It's true that deconvolving adds time and data volume and especially cost, but in trade you get an image that is substantially sharper, with reduced noise and background, and more quantitatively accurate*. Regarding whether to just go with a point scanning confocal, I don't see it as a simple question of better or worse**. A nuclear-cytoplasmic translocation assay with monolayer cells works just as well on a widefield, and (in my experience!) many types of biosensor assay work better with a properly set up widefield. The 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cameras have by far the best acquisition speeds. I don't know whether widefields still have a more linear relationship between sample brightness and detected signal, but the last time I checked that was still true. (*) Deconvolution is quantitatively useful as long as people make sure to tell the software to preserve the original intensity values. One of my complaints about Hyvolution was that you could not do that, so I just used the Huygens package that came with it. I don't know whether Lightning gives you that option...if not then caveat emptor. (**) My advice mostly applies to turnkey stuff that any lab can implement, not exotic techniques available to folks with specialists or engineers on hand. Best, T Timothy Feinstein, Ph.D. esearch Scientist Department of Developmental Biology University of Pittsburgh On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <[hidden email] on behalf of [hidden email]> wrote: I'll point out, that you can, of course, deconvolve confocal images too. So, while you can indeed get near confocal quality with well-acquired wf data after deconvolution, you can also get near SR quality when deconvolving a well-acquired stack from a confocal. And then you can also deconvolve a SR stack and get... well you get the idea! It's like an arms race. I have the Hyvolution and had access for a couple of weeks to the Lighting, and now confocal images look blurry to me. Avi -- Avi Jacob, Ph.D. Kanbar Light Microscopy Unit The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 529002, Israel On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=efIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc%3D&reserved=0 > Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=MNM8i3iDaQuMA9LF766%2FyvCyp94jmie4IaC5qIEWclA%3D&reserved=0 and include the link in your posting. > ***** > > Hi Mika, > > White et al 1987 ( https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fjcb.rupress.org%2Fcontent%2F105%2F1%2F41.long&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=a6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0%3D&reserved=0 ) made a > compelling case for point scanning confocal microscopes: collect just > the in focus light with instant gratification. The case has not changed, > the hardware (especially data deluge side) has gotten a lot better. I > note that both widefield detectors and PMT/APD/hybrid detectors/others > have gotten a lot better in the 32 years from 1987! As have the optics > and automation. > > Paul Goodwin 2014 ( https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24974028&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=GDve8BuxhLDAdBPKywRQoMTYV%2BqSk9aMcz2WrZCHPU8%3D&reserved=0 ) made > a nice case for quantitative deconvolution microscopy (and on very high > quality specimens, ~10% improvement in resolution compared to simple > widefield), but historically slow. > > Now, with 'instant gratification' spatial deconvolution, thanks to the > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > ... not including the deconvolution software module price), widefield, > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > about the price point of recent fiber lasers, which could become much > better price if achieve 'economy of scale'), and of course, point > scanning confocal microscopes. > > Spatial deconvolution (especially if someone $uccessfully implements > joint spatial deconvolution and spectral unmixing, multiple cameras - > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > really single molecule counting (and DNA-PAINT eliminates the classic > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > More significantly, DNA-PAINT and related methods (single molecule RNA > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > multiplex -- with single molecule counting -- to whatever plex is needed > to answer the 'biological question(s)' being posed. > > All that said, the installed base of research grade point scanning > confocal microscopes is large (5000+) and efficient at acquiring high > quality images, to the point that user's sample preparation (and > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > is much more limiting than the microscopes. > > George > > p.s. a couple of references not included in above: > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy > second edition https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Flink.springer.com%2Fbook%2F10.1007%252F978-3-319-97454-5&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=%2FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc%2BnO2gdk60Us%3D&reserved=0 > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41596-018-0117-3&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=CF%2Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw%3D&reserved=0 > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.mdpi.com%2F2073-4425%2F9%2F12%2F621&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=iwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM%3D&reserved=0 > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > with $1550 CMOS cameras) ... > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41598-018-19981-z&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257860747&sdata=wN%2FHP30wNUE0%2BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c%3D&reserved=0 > > Moffitt et al 2018 ... > https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2Feaau5324.long&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=FR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w%3D&reserved=0 and > commentary https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2F749&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=o0B379uuh%2FrB6gS9n6lG%2BjAZeztiYHZVmICwX4ghKh8%3D&reserved=0 > > *** > > Some resolution numbers: > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength > / NA (I routinely drop the 0.61 from 0.61) > > 0.6 * 500 / 1.4 = 214 nm > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > pixel size matched or interpolate optimally). > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > resolution wrt 1 and smaller pinhole size, source of the values below, > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput > (which doesn't matter if target is photostable). > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > FastAiryScan). > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > Most modern point scanning confocal microscopes have a 405 nm laser, so > if using BV421 (and ignoring potential photobleaching for a moment), > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > or in reflection mode, i.e. nanodiamond or nanogold, > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > photobleaching, so infinite number of photons (though also no blinking, > so not usually eligible for precision localization) ... > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > and not going completely exotic with NA (i.e. 1.65), if perfectly > refractive index match with a fairly conventional 1.49 NA lens, and > inreflectance: > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to > 101 nm. > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out > of disk space or time or money), since precision localization is > resolution divided by square root of number of photons, ex. 250 nm XY > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > photons per target further, but why bother? > > *** > > point scanning confocal microscopes are also great platforms for > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F18387308&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=ZQTosrSoPFpCa8Y3IdEa9Xz%2B4RHC8JO4gBppHmzkazo%3D&reserved=0 > > *** > > p.p.s. Disclosures I am ... > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > 3/18/2019 afternoon) ... see pdf download page, > > THUNDER Technology Note > THUNDER Imagers: How Do They Really Work? > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.leica-microsystems.com%2Fscience-lab%2Fthunder-technology-note&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=auRUORuEKAir87%2Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM%3D&reserved=0 > > 2. hosting Nikon confocal demos in May 2019. > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > summer. > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F28261321&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=uVw0kiW1baZV0RJv%2Bk1ObKznhw51pemnCMehjHkUa2g%3D&reserved=0 > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=dHOCjeBda4sSZPf%2FB2%2B5Sv3Q8Qzcs708p4we8vHf%2FIg%3D&reserved=0 > > Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Ctnf8%40PITT.EDU%7Ce2895884dda94858a22408d6ab8114ff%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636884968257870756&sdata=cnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg%3D&reserved=0 and include the link in your > posting. > > ***** > > > > Hi. > > There are software solutions that are able to create image data from > wide-field and even microscope systems with seemingly similar quality to > that obtained from confocal systems. > > > > The comparison of acquisition vs. software is essentially a comparison > of image acquisition vs. image processing. While a software solution is way > cheaper than a hardware solution if it is able to produce image data with > equal quality why would anyone choose to invest to a confocal anymore? > > > > I’m looking forward to a vidid discussion! > > > >> m > |
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Alison J. North |
Re: General question: Software vs. hardware
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, So this is a very timely discussion because I have been discussing with my staff whether there are data sets that should NOT be deconvolved, and if so, how does one decide that? I too attended Jim Pawley's wonderful course (he was certainly a huge character as well as an incredibly fantastic microscopist, and he will certainly be remembered by all!), so I have generally worked under the assumption that one should deconvolve all confocal data. But I am also very aware of the potential for artifacts if a data set isn't "good enough" for deconvolution. Obviously the ideal situation is to acquire an optimal data set - well-prepared sample, bright staining, Nyquist sampling etc. etc., and a high S:N ratio - and by sticking to these rules, our deconvolved DeltaVision images or confocal images of fixed samples have always looked great. But nowadays we are faced with different scenarios, particularly when you are attempting to do very rapid imaging of live, weakly expressing cells, while attempting to minimize phototoxicity. In that case you can end up with pretty lousy S:N ratios, because maintaining cell viability or imaging fast enough is more critical. For example, I have a lovely new iSIM in my lab, for which the initial resolution increase is achieved by the hardware, but the second step in resolution increase is via deconvolution. The whole point of this instrument is for rapid, super-resolution imaging - so we can't simply increase exposure times to improve S:N, turning up the laser power will obviously kill the cells, and we can't increase the pixel size or we'll lose the resolution. And I assume a lot of the new types of super-resolution instrument out there must leave you facing the same issue, since live cell imaging invariably forces you to compromise somewhere within the imaging triangle (or hexagon, or whatever we've got up to now!). Therefore my question is, are there papers out there which have compared deconvolution algorithms and looked at the potential for artifacts on really low S:N images, which we could use to advise our researchers on what is the minimum you can get away with before you really shouldn't be deconvolving the data set at all? Also, are there papers showing the effect of undersampling in the z-axis on the resulting deconvolved images (as is often the case on our spinning disk system)? I haven't managed to find any yet (though I confess I've been too busy with other stuff to spend too many hours searching!), so if anybody could point me to some good references I'd be most grateful. I have spoken with several renowned microscopists about whether deconvolution is always a good idea under such circumstances, and the gut reaction appears to be no, but I could do with some hard and fast validation for teaching purposes. Many thanks in advance! Alison On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=vfjahhKueherhfphxmr-Smw_FhB4QUlg-FsBATM1kl0&e= > Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=edOjY0NZEIhVq-zWzgNHHDaTrutHrPj1Rl6rcwE_B4M&e= and include the link in your posting. > ***** > > Avi, I really agree with your point. I feel that people to deconvolve any time spatial information is critical, whether they're using widefield, CLSM, spinning disc, or light sheet. It's true that deconvolving adds time and data volume and especially cost, but in trade you get an image that is substantially sharper, with reduced noise and background, and more quantitatively accurate*. > > Regarding whether to just go with a point scanning confocal, I don't see it as a simple question of better or worse**. A nuclear-cytoplasmic translocation assay with monolayer cells works just as well on a widefield, and (in my experience!) many types of biosensor assay work better with a properly set up widefield. The 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cameras have by far the best acquisition speeds. I don't know whether widefields still have a more linear relationship between sample brightness and detected signal, but the last time I checked that was still true. > > (*) Deconvolution is quantitatively useful as long as people make sure to tell the software to preserve the original intensity values. One of my complaints about Hyvolution was that you could not do that, so I just used the Huygens package that came with it. I don't know whether Lightning gives you that option...if not then caveat emptor. > > (**) My advice mostly applies to turnkey stuff that any lab can implement, not exotic techniques available to folks with specialists or engineers on hand. > > Best, > > > T > Timothy Feinstein, Ph.D. esearch Scientist > Department of Developmental Biology > University of Pittsburgh > > > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <[hidden email] on behalf of [hidden email]> wrote: > > > I'll point out, that you can, of course, deconvolve confocal images too. > So, while you can indeed get near confocal quality with well-acquired wf > data after deconvolution, you can also get near SR quality when > deconvolving a well-acquired stack from a confocal. And then you can also > deconvolve a SR stack and get... well you get the idea! It's like an arms > race. > I have the Hyvolution and had access for a couple of weeks to the Lighting, > and now confocal images look blurry to me. > Avi > > -- > Avi Jacob, Ph.D. > Kanbar Light Microscopy Unit > The Mina & Everard Goodman Faculty of Life Sciences > Bar-Ilan University, Ramat-Gan 529002, Israel > > > > On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DefIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=9Uk5ZZzc8LP7V7Oj2aFSRbVluY0mpE-7XPLYdsyviMk&e= > > Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=Gy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M&e= and include the link in your posting. > > ***** > > > > Hi Mika, > > > > White et al 1987 ( https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4&e= ) made a > > compelling case for point scanning confocal microscopes: collect just > > the in focus light with instant gratification. The case has not changed, > > the hardware (especially data deluge side) has gotten a lot better. I > > note that both widefield detectors and PMT/APD/hybrid detectors/others > > have gotten a lot better in the 32 years from 1987! As have the optics > > and automation. > > > > Paul Goodwin 2014 ( https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=PZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84&e= ) made > > a nice case for quantitative deconvolution microscopy (and on very high > > quality specimens, ~10% improvement in resolution compared to simple > > widefield), but historically slow. > > > > Now, with 'instant gratification' spatial deconvolution, thanks to the > > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > > ... not including the deconvolution software module price), widefield, > > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > > about the price point of recent fiber lasers, which could become much > > better price if achieve 'economy of scale'), and of course, point > > scanning confocal microscopes. > > > > Spatial deconvolution (especially if someone $uccessfully implements > > joint spatial deconvolution and spectral unmixing, multiple cameras - > > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > > really single molecule counting (and DNA-PAINT eliminates the classic > > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > > > More significantly, DNA-PAINT and related methods (single molecule RNA > > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > > multiplex -- with single molecule counting -- to whatever plex is needed > > to answer the 'biological question(s)' being posed. > > > > All that said, the installed base of research grade point scanning > > confocal microscopes is large (5000+) and efficient at acquiring high > > quality images, to the point that user's sample preparation (and > > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > > is much more limiting than the microscopes. > > > > George > > > > p.s. a couple of references not included in above: > > > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy > > second edition https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE&e= > > > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=YeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU&e= > > > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=tJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE&e= > > > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > > with $1550 CMOS cameras) ... > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=TIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM&e= > > > > Moffitt et al 2018 ... > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0&e= and > > commentary https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=XeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ&e= > > > > *** > > > > Some resolution numbers: > > > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength > > / NA (I routinely drop the 0.61 from 0.61) > > > > 0.6 * 500 / 1.4 = 214 nm > > > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > > pixel size matched or interpolate optimally). > > > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles > > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > > resolution wrt 1 and smaller pinhole size, source of the values below, > > > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput > > (which doesn't matter if target is photostable). > > > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > > FastAiryScan). > > > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > > > Most modern point scanning confocal microscopes have a 405 nm laser, so > > if using BV421 (and ignoring potential photobleaching for a moment), > > > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > > > or in reflection mode, i.e. nanodiamond or nanogold, > > > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > > photobleaching, so infinite number of photons (though also no blinking, > > so not usually eligible for precision localization) ... > > > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > > > and not going completely exotic with NA (i.e. 1.65), if perfectly > > refractive index match with a fairly conventional 1.49 NA lens, and > > inreflectance: > > > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to > > 101 nm. > > > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out > > of disk space or time or money), since precision localization is > > resolution divided by square root of number of photons, ex. 250 nm XY > > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > > photons per target further, but why bother? > > > > *** > > > > point scanning confocal microscopes are also great platforms for > > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=meRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw&e= > > > > *** > > > > p.p.s. Disclosures I am ... > > > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > > 3/18/2019 afternoon) ... see pdf download page, > > > > THUNDER Technology Note > > THUNDER Imagers: How Do They Really Work? > > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=LmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco&e= > > > > 2. hosting Nikon confocal demos in May 2019. > > > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > > summer. > > > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=KC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM&e= > > > > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=qZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE&e= > > > Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=jqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw&s=YzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU&e= and include the link in your > > posting. > > > ***** > > > > > > Hi. > > > There are software solutions that are able to create image data from > > wide-field and even microscope systems with seemingly similar quality to > > that obtained from confocal systems. > > > > > > The comparison of acquisition vs. software is essentially a comparison > > of image acquisition vs. image processing. While a software solution is way > > cheaper than a hardware solution if it is able to produce image data with > > equal quality why would anyone choose to invest to a confocal anymore? > > > > > > I’m looking forward to a vidid discussion! > > > > > >> m > > > > Alison J. North, Ph.D., Research Associate Professor and Senior 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 |
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Nicolai.Urban@mpfi.org |
Re: General question: Software vs. hardware
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In reply to this post by Tim Feinstein
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi everyone, I agree with most that has been said. I am firmly of the belief that you should try to acquire the best possible images and then you can always improve them further by using deconvolution or other methods. The more imaging artifacts and erroneous light you can reject during the imaging process, the less you will have to deal with later. Also, many journals have started asking about providing the raw data for figures as well - for which having superior data right off the bat is a huge benefit. One large risk of using software is that you can incur all sorts of artifacts, especially if you do not know what you are doing or if you push the limits of the deconvolution too far. I have had to gently let down some scientists who were excited about seeing this or that in their deconvolved data, with the raw data simply not supporting it. Furthermore, as Timothy pointed out, some deconvolution software automatically applies certain procedures or maybe does not make very clear exactly what has been done - reiterating the statement: you should know what you are doing when using software to improve your images. (Or at least consult with someone who does) So in conclusion: get the best images you can, and then improve them even further. The results speak for themselves. Best, Nicolai -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Feinstein, Timothy N Sent: Montag, 18. März 2019 09:56 To: [hidden email] Subject: Re: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988468333&sdata=%2B0sr4LxFc4S9c9F6hK8DeKlbHShPwXlrKWzWgWTUp7Y%3D&reserved=0 Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting. ***** Avi, I really agree with your point. I feel that people to deconvolve any time spatial information is critical, whether they're using widefield, CLSM, spinning disc, or light sheet. It's true that deconvolving adds time and data volume and especially cost, but in trade you get an image that is substantially sharper, with reduced noise and background, and more quantitatively accurate*. Regarding whether to just go with a point scanning confocal, I don't see it as a simple question of better or worse**. A nuclear-cytoplasmic translocation assay with monolayer cells works just as well on a widefield, and (in my experience!) many types of biosensor assay work better with a properly set up widefield. The 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cameras have by far the best acquisition speeds. I don't know whether widefields still have a more linear relationship between sample brightness and detected signal, but the last time I checked that was still true. (*) Deconvolution is quantitatively useful as long as people make sure to tell the software to preserve the original intensity values. One of my complaints about Hyvolution was that you could not do that, so I just used the Huygens package that came with it. I don't know whether Lightning gives you that option...if not then caveat emptor. (**) My advice mostly applies to turnkey stuff that any lab can implement, not exotic techniques available to folks with specialists or engineers on hand. Best, T Timothy Feinstein, Ph.D. esearch Scientist Department of Developmental Biology University of Pittsburgh On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <[hidden email] on behalf of [hidden email]> wrote: I'll point out, that you can, of course, deconvolve confocal images too. So, while you can indeed get near confocal quality with well-acquired wf data after deconvolution, you can also get near SR quality when deconvolving a well-acquired stack from a confocal. And then you can also deconvolve a SR stack and get... well you get the idea! It's like an arms race. I have the Hyvolution and had access for a couple of weeks to the Lighting, and now confocal images look blurry to me. Avi -- Avi Jacob, Ph.D. Kanbar Light Microscopy Unit The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 529002, Israel On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=8UtAwuTn3RvnsxIPIRQhzw%2BYhqpEwpdVbRUgT4i2v0Q%3D&reserved=0 > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting. > ***** > > Hi Mika, > > White et al 1987 ( https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fjcb.rupress.org%2Fcontent%2F105%2F1%2F41.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=nzaoZX0omJibqA4g7XfhlKbeiTPdqXNnUtAstnMp%2F7s%3D&reserved=0 ) made a > compelling case for point scanning confocal microscopes: collect just > the in focus light with instant gratification. The case has not changed, > the hardware (especially data deluge side) has gotten a lot better. I > note that both widefield detectors and PMT/APD/hybrid detectors/others > have gotten a lot better in the 32 years from 1987! As have the optics > and automation. > > Paul Goodwin 2014 ( https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24974028&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=IJYfSZDn9%2B8IqtOVPtr4oHIsVQ0DwNU%2FcYDBWHLCtls%3D&reserved=0 ) made > a nice case for quantitative deconvolution microscopy (and on very high > quality specimens, ~10% improvement in resolution compared to simple > widefield), but historically slow. > > Now, with 'instant gratification' spatial deconvolution, thanks to the > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > ... not including the deconvolution software module price), widefield, > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > about the price point of recent fiber lasers, which could become much > better price if achieve 'economy of scale'), and of course, point > scanning confocal microscopes. > > Spatial deconvolution (especially if someone $uccessfully implements > joint spatial deconvolution and spectral unmixing, multiple cameras - > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > really single molecule counting (and DNA-PAINT eliminates the classic > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > More significantly, DNA-PAINT and related methods (single molecule RNA > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > multiplex -- with single molecule counting -- to whatever plex is needed > to answer the 'biological question(s)' being posed. > > All that said, the installed base of research grade point scanning > confocal microscopes is large (5000+) and efficient at acquiring high > quality images, to the point that user's sample preparation (and > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > is much more limiting than the microscopes. > > George > > p.s. a couple of references not included in above: > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy > second edition https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Flink.springer.com%2Fbook%2F10.1007%252F978-3-319-97454-5&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=sNoUx5yoXFfjFCPvziQDweJ2PJXfRhOtB0Tg0rWbj04%3D&reserved=0 > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41596-018-0117-3&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=4WrXBtCidTZfrARUzRcm1UvolmzLg1pG0SZkErLU93E%3D&reserved=0 > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.mdpi.com%2F2073-4425%2F9%2F12%2F621&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=VW4WoAKHnc2hhYQEgPUqt%2BeFdN%2Bw7fADCLXdgDoU98k%3D&reserved=0 > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > with $1550 CMOS cameras) ... > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41598-018-19981-z&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=zxdHdhYM6%2BaCes%2Bq2NoF70EmKSiwOvmHrfW%2BRuSjClA%3D&reserved=0 > > Moffitt et al 2018 ... > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2Feaau5324.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=z42w%2Fn2NVAWdTD5q7mUrwhOliTRDW4%2BCZQgAEPvnhTM%3D&reserved=0 and > commentary https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2F749&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=FgPwqbrQ66L398R0tyiOD%2BTrJovMlb22NCmpnV%2BytgQ%3D&reserved=0 > > *** > > Some resolution numbers: > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength > / NA (I routinely drop the 0.61 from 0.61) > > 0.6 * 500 / 1.4 = 214 nm > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > pixel size matched or interpolate optimally). > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > resolution wrt 1 and smaller pinhole size, source of the values below, > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput > (which doesn't matter if target is photostable). > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > FastAiryScan). > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > Most modern point scanning confocal microscopes have a 405 nm laser, so > if using BV421 (and ignoring potential photobleaching for a moment), > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > or in reflection mode, i.e. nanodiamond or nanogold, > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > photobleaching, so infinite number of photons (though also no blinking, > so not usually eligible for precision localization) ... > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > and not going completely exotic with NA (i.e. 1.65), if perfectly > refractive index match with a fairly conventional 1.49 NA lens, and > inreflectance: > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to > 101 nm. > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out > of disk space or time or money), since precision localization is > resolution divided by square root of number of photons, ex. 250 nm XY > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > photons per target further, but why bother? > > *** > > point scanning confocal microscopes are also great platforms for > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F18387308&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=8L6sZRJKBP%2Fp%2FZm5YjGAID6uR8sprK8yKE5jSTIOZes%3D&reserved=0 > > *** > > p.p.s. Disclosures I am ... > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > 3/18/2019 afternoon) ... see pdf download page, > > THUNDER Technology Note > THUNDER Imagers: How Do They Really Work? > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.leica-microsystems.com%2Fscience-lab%2Fthunder-technology-note&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=XZDsAx4PBnd7K5BF9dshnIrDk6glSie%2BQZA%2FmvDCBRA%3D&reserved=0 > > 2. hosting Nikon confocal demos in May 2019. > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > summer. > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F28261321&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=2oiWeA2uoGgy3GrBSjVSiEr%2FOgV3pYlJ43MHOOD6FPg%3D&reserved=0 > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=5aNe3XPrrvZ2lpSR2%2F1K1ereIMtgE3hpSF1r3HNaaFU%3D&reserved=0 > > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=PmBfP4DRz%2FvetYmcPIr%2BZDuWIUNmbZaetHNkQ8Wg%2BU4%3D&reserved=0 and include the link in your > posting. > > ***** > > > > Hi. > > There are software solutions that are able to create image data from > wide-field and even microscope systems with seemingly similar quality to > that obtained from confocal systems. > > > > The comparison of acquisition vs. software is essentially a comparison > of image acquisition vs. image processing. While a software solution is way > cheaper than a hardware solution if it is able to produce image data with > equal quality why would anyone choose to invest to a confocal anymore? > > > > I’m looking forward to a vidid discussion! > > > >> m > |
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F Javier Diez Guerra |
Re: General question: Software vs. hardware
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, Very interesting discussion! Most opinions gather around the idea of rely on hardware and make use of software as much as you can. But, not strictly speaking on deconvolution, I would be interested to listen also to opinions on more blurred mixtures of hardware and software, such as structured illumination (SIM) in their latest implementations, for example Lattice SIM. Best, Javier El 18/03/2019 a las 16:11, [hidden email] escribió: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi everyone, > > I agree with most that has been said. I am firmly of the belief that you should try to acquire the best possible images and then you can always improve them further by using deconvolution or other methods. The more imaging artifacts and erroneous light you can reject during the imaging process, the less you will have to deal with later. Also, many journals have started asking about providing the raw data for figures as well - for which having superior data right off the bat is a huge benefit. > > One large risk of using software is that you can incur all sorts of artifacts, especially if you do not know what you are doing or if you push the limits of the deconvolution too far. I have had to gently let down some scientists who were excited about seeing this or that in their deconvolved data, with the raw data simply not supporting it. Furthermore, as Timothy pointed out, some deconvolution software automatically applies certain procedures or maybe does not make very clear exactly what has been done - reiterating the statement: you should know what you are doing when using software to improve your images. (Or at least consult with someone who does) > > So in conclusion: get the best images you can, and then improve them even further. The results speak for themselves. > > Best, > Nicolai > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On Behalf Of Feinstein, Timothy N > Sent: Montag, 18. März 2019 09:56 > To: [hidden email] > Subject: Re: General question: Software vs. hardware > > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988468333&sdata=%2B0sr4LxFc4S9c9F6hK8DeKlbHShPwXlrKWzWgWTUp7Y%3D&reserved=0 > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting. > ***** > > Avi, I really agree with your point. I feel that people to deconvolve any time spatial information is critical, whether they're using widefield, CLSM, spinning disc, or light sheet. It's true that deconvolving adds time and data volume and especially cost, but in trade you get an image that is substantially sharper, with reduced noise and background, and more quantitatively accurate*. > > Regarding whether to just go with a point scanning confocal, I don't see it as a simple question of better or worse**. A nuclear-cytoplasmic translocation assay with monolayer cells works just as well on a widefield, and (in my experience!) many types of biosensor assay work better with a properly set up widefield. The 16-bit depth of widefield images is nice for quantitation, and modern sCMOS cameras have by far the best acquisition speeds. I don't know whether widefields still have a more linear relationship between sample brightness and detected signal, but the last time I checked that was still true. > > (*) Deconvolution is quantitatively useful as long as people make sure to tell the software to preserve the original intensity values. One of my complaints about Hyvolution was that you could not do that, so I just used the Huygens package that came with it. I don't know whether Lightning gives you that option...if not then caveat emptor. > > (**) My advice mostly applies to turnkey stuff that any lab can implement, not exotic techniques available to folks with specialists or engineers on hand. > > Best, > > > T > Timothy Feinstein, Ph.D. esearch Scientist Department of Developmental Biology University of Pittsburgh > > > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi Jacob" <[hidden email] on behalf of [hidden email]> wrote: > > > I'll point out, that you can, of course, deconvolve confocal images too. > So, while you can indeed get near confocal quality with well-acquired wf > data after deconvolution, you can also get near SR quality when > deconvolving a well-acquired stack from a confocal. And then you can also > deconvolve a SR stack and get... well you get the idea! It's like an arms > race. > I have the Hyvolution and had access for a couple of weeks to the Lighting, > and now confocal images look blurry to me. > Avi > > -- > Avi Jacob, Ph.D. > Kanbar Light Microscopy Unit > The Mina & Everard Goodman Faculty of Life Sciences > Bar-Ilan University, Ramat-Gan 529002, Israel > > > > On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> > wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=8UtAwuTn3RvnsxIPIRQhzw%2BYhqpEwpdVbRUgT4i2v0Q%3D&reserved=0 > > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=7oaSK53PubjjY5%2Fsqe8ifzN8JxQsnG6rCZ07bJj2X2A%3D&reserved=0 and include the link in your posting. > > ***** > > > > Hi Mika, > > > > White et al 1987 ( https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fjcb.rupress.org%2Fcontent%2F105%2F1%2F41.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=nzaoZX0omJibqA4g7XfhlKbeiTPdqXNnUtAstnMp%2F7s%3D&reserved=0 ) made a > > compelling case for point scanning confocal microscopes: collect just > > the in focus light with instant gratification. The case has not changed, > > the hardware (especially data deluge side) has gotten a lot better. I > > note that both widefield detectors and PMT/APD/hybrid detectors/others > > have gotten a lot better in the 32 years from 1987! As have the optics > > and automation. > > > > Paul Goodwin 2014 ( https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F24974028&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=IJYfSZDn9%2B8IqtOVPtr4oHIsVQ0DwNU%2FcYDBWHLCtls%3D&reserved=0 ) made > > a nice case for quantitative deconvolution microscopy (and on very high > > quality specimens, ~10% improvement in resolution compared to simple > > widefield), but historically slow. > > > > Now, with 'instant gratification' spatial deconvolution, thanks to the > > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > > ... not including the deconvolution software module price), widefield, > > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > > about the price point of recent fiber lasers, which could become much > > better price if achieve 'economy of scale'), and of course, point > > scanning confocal microscopes. > > > > Spatial deconvolution (especially if someone $uccessfully implements > > joint spatial deconvolution and spectral unmixing, multiple cameras - > > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > > really single molecule counting (and DNA-PAINT eliminates the classic > > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > > > More significantly, DNA-PAINT and related methods (single molecule RNA > > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > > multiplex -- with single molecule counting -- to whatever plex is needed > > to answer the 'biological question(s)' being posed. > > > > All that said, the installed base of research grade point scanning > > confocal microscopes is large (5000+) and efficient at acquiring high > > quality images, to the point that user's sample preparation (and > > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > > is much more limiting than the microscopes. > > > > George > > > > p.s. a couple of references not included in above: > > > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal Microscopy > > second edition https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Flink.springer.com%2Fbook%2F10.1007%252F978-3-319-97454-5&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=sNoUx5yoXFfjFCPvziQDweJ2PJXfRhOtB0Tg0rWbj04%3D&reserved=0 > > > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41596-018-0117-3&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=4WrXBtCidTZfrARUzRcm1UvolmzLg1pG0SZkErLU93E%3D&reserved=0 > > > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.mdpi.com%2F2073-4425%2F9%2F12%2F621&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988478337&sdata=VW4WoAKHnc2hhYQEgPUqt%2BeFdN%2Bw7fADCLXdgDoU98k%3D&reserved=0 > > > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > > with $1550 CMOS cameras) ... > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.nature.com%2Farticles%2Fs41598-018-19981-z&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=zxdHdhYM6%2BaCes%2Bq2NoF70EmKSiwOvmHrfW%2BRuSjClA%3D&reserved=0 > > > > Moffitt et al 2018 ... > > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2Feaau5324.long&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=z42w%2Fn2NVAWdTD5q7mUrwhOliTRDW4%2BCZQgAEPvnhTM%3D&reserved=0 and > > commentary https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fscience.sciencemag.org%2Fcontent%2F362%2F6416%2F749&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=FgPwqbrQ66L398R0tyiOD%2BTrJovMlb22NCmpnV%2BytgQ%3D&reserved=0 > > > > *** > > > > Some resolution numbers: > > > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * wqavelength > > / NA (I routinely drop the 0.61 from 0.61) > > > > 0.6 * 500 / 1.4 = 214 nm > > > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > > pixel size matched or interpolate optimally). > > > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 Principles > > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > > resolution wrt 1 and smaller pinhole size, source of the values below, > > > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons throughput > > (which doesn't matter if target is photostable). > > > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > > FastAiryScan). > > > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > > > Most modern point scanning confocal microscopes have a 405 nm laser, so > > if using BV421 (and ignoring potential photobleaching for a moment), > > > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > > > or in reflection mode, i.e. nanodiamond or nanogold, > > > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > > photobleaching, so infinite number of photons (though also no blinking, > > so not usually eligible for precision localization) ... > > > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > > > and not going completely exotic with NA (i.e. 1.65), if perfectly > > refractive index match with a fairly conventional 1.49 NA lens, and > > inreflectance: > > > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 to > > 101 nm. > > > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run out > > of disk space or time or money), since precision localization is > > resolution divided by square root of number of photons, ex. 250 nm XY > > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > > photons per target further, but why bother? > > > > *** > > > > point scanning confocal microscopes are also great platforms for > > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F18387308&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=8L6sZRJKBP%2Fp%2FZm5YjGAID6uR8sprK8yKE5jSTIOZes%3D&reserved=0 > > > > *** > > > > p.p.s. Disclosures I am ... > > > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > > 3/18/2019 afternoon) ... see pdf download page, > > > > THUNDER Technology Note > > THUNDER Imagers: How Do They Really Work? > > > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.leica-microsystems.com%2Fscience-lab%2Fthunder-technology-note&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=XZDsAx4PBnd7K5BF9dshnIrDk6glSie%2BQZA%2FmvDCBRA%3D&reserved=0 > > > > 2. hosting Nikon confocal demos in May 2019. > > > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > > summer. > > > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > > https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpubmed%2F28261321&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=2oiWeA2uoGgy3GrBSjVSiEr%2FOgV3pYlJ43MHOOD6FPg%3D&reserved=0 > > > > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=5aNe3XPrrvZ2lpSR2%2F1K1ereIMtgE3hpSF1r3HNaaFU%3D&reserved=0 > > > Post images on https://nam03.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C89f6194738774f020d2f08d6aba987cd%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636885141988488345&sdata=PmBfP4DRz%2FvetYmcPIr%2BZDuWIUNmbZaetHNkQ8Wg%2BU4%3D&reserved=0 and include the link in your > > posting. > > > ***** > > > > > > Hi. > > > There are software solutions that are able to create image data from > > wide-field and even microscope systems with seemingly similar quality to > > that obtained from confocal systems. > > > > > > The comparison of acquisition vs. software is essentially a comparison > > of image acquisition vs. image processing. While a software solution is way > > cheaper than a hardware solution if it is able to produce image data with > > equal quality why would anyone choose to invest to a confocal anymore? > > > > > > I’m looking forward to a vidid discussion! > > > > > >> m > > > > |
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Steffen Dietzel |
Re: General question: Software vs. hardware
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In reply to this post by ICIT
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Dear Mika, it would be really helpful if you could be more specific. What exactly do you want to compare? There are so many options out there theses days that it is difficult to guess what you mean. Having said that, as Avi pointed out, it is generally best to first get a good image by physical means and then do the computational improvement like deconvolution. That is true for widefield, confocal and also STED superresolution. So, confocal and decon is better than confocal alone or widefield and decon. Although for some applications widefield and decon might be good enough. Steffen Am 17.03.2019 um 16:17 schrieb Mika Ruonala: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Essentially, this is the question. > -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany http://www.bioimaging.bmc.med.uni-muenchen.de |
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Cammer, Michael |
Re: General question: Software vs. hardware
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In reply to this post by Avi Jacob
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Many applications overlap but here are three examples of when laser scanning confocal is indispensable: You cannot tell by widefield whether you are seeing a fluorescent labeled structure or reflection from light emitted by another bright area. Some thick tissues. Some of the weird chambers brought in by engineers, transwell chambers, and other oddities. Perhaps this does not really address the original question, but we have found that where deconvolution requires sitting at another computer with another software package and paying a core a fee for use, it just isn’t going to happen. Cheers- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 C: 914-309-3270 [hidden email] http://nyulmc.org/micros http://microscopynotes.com/ -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Avi Jacob Sent: Monday, March 18, 2019 4:59 AM To: [hidden email] Subject: Re: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=3LqjIRKGooGbWufKQIfCOyBhknMhzM4msy9LD6YtGwo&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=LwlU9Ttd8-NvvVIj2xG11Fkz5KupvCoj80DexAghEHY&e= and include the link in your posting. ***** I'll point out, that you can, of course, deconvolve confocal images too. So, while you can indeed get near confocal quality with well-acquired wf data after deconvolution, you can also get near SR quality when deconvolving a well-acquired stack from a confocal. And then you can also deconvolve a SR stack and get... well you get the idea! It's like an arms race. I have the Hyvolution and had access for a couple of weeks to the Lighting, and now confocal images look blurry to me. Avi -- Avi Jacob, Ph.D. Kanbar Light Microscopy Unit The Mina & Everard Goodman Faculty of Life Sciences Bar-Ilan University, Ramat-Gan 5290002, Israel On Sun, Mar 17, 2019 at 6:04 PM George McNamara <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi- > 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeEl > Zfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=Qh > UrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=3LqjIRKGooGbWufKQIfCOyBhkn > MhzM4msy9LD6YtGwo&e= Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=LwlU9Ttd8-NvvVIj2xG11Fkz5KupvCoj80DexAghEHY&e= and include the link in your posting. > ***** > > Hi Mika, > > White et al 1987 ( > https://urldefense.proofpoint.com/v2/url?u=http-3A__jcb.rupress.org_co > ntent_105_1_41.long&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII > ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_ > r1_9otpqB337-xfUGpGNb7ONvc&s=yVMwIyG9dOwJekwvrhAd-YcpXuwUz0br0S_5PdDo0 > 6A&e= ) made a compelling case for point scanning confocal > microscopes: collect just the in focus light with instant gratification. The case has not changed, the hardware (especially data deluge side) has gotten a lot better. I note that both widefield detectors and PMT/APD/hybrid detectors/others have gotten a lot better in the 32 years from 1987! As have the optics and automation. > > Paul Goodwin 2014 ( > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih. > gov_pubmed_24974028&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII > ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=Ua3LXNZyL2gL-lg5mxuXVCNKXwSMTo41DbEEcPPLBiQ&e= ) made a nice case for quantitative deconvolution microscopy (and on very high quality specimens, ~10% improvement in resolution compared to simple widefield), but historically slow. > > Now, with 'instant gratification' spatial deconvolution, thanks to the > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb ram, $2500 > ... not including the deconvolution software module price), widefield, > spinning disk (and slightly exotic variants like iSIM, DMD based, etc, > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm excited > about the price point of recent fiber lasers, which could become much > better price if achieve 'economy of scale'), and of course, point > scanning confocal microscopes. > > Spatial deconvolution (especially if someone $uccessfully implements > joint spatial deconvolution and spectral unmixing, multiple cameras - > for 4 cameras see Babcock 2018, mentions aiming for 8 cameras) helps > with Expansion Microscopy and/or DNA-PAINT, to go super-resolution ... > really single molecule counting (and DNA-PAINT eliminates the classic > issue of PALM/STORM/FPALM of not counting every molecule). Sure, > DNA-PAINT (like STORM etc) have the issue of a whole lotta images > acquired. Data deluge: who cares? Jerome & Price's 10th commandment of > confocal imaging is: "10. Storage Media Is Essentially Free and Infinite". > > More significantly, DNA-PAINT and related methods (single molecule RNA > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) also enable > multiplex -- with single molecule counting -- to whatever plex is > needed to answer the 'biological question(s)' being posed. > > All that said, the installed base of research grade point scanning > confocal microscopes is large (5000+) and efficient at acquiring high > quality images, to the point that user's sample preparation (and > avoidance of purchasing stuff from 'Santa Crap' and similar companies) > is much more limiting than the microscopes. > > George > > p.s. a couple of references not included in above: > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal > Microscopy second edition > https://urldefense.proofpoint.com/v2/url?u=https-3A__link.springer.com > _book_10.1007-252F978-2D3-2D319-2D97454-2D5&d=DwIFaQ&c=j5oPpO0eBH1iio4 > 8DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0Rkn > E5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=OLwJ4Tn_dtindX_od > PzsUb7FqLW0x-HCs7F8Mb3VwyA&e= > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar > ticles_s41596-2D018-2D0117-2D3&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc0 > 4rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmP > qL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=cUzsjl7SIzMQUNgwgKER9mDPZEMohi > 5rhFZJwOnal6s&e= > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.mdpi.com_2073 > -2D4425_9_12_621&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuC > s&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_ > 9otpqB337-xfUGpGNb7ONvc&s=oaz40_UfNUr03mxrG9Wo1FC400X08izokzftEHhlf9w& > e= > > Babcock 2018 (4 --> 8 cameras, single molecule localization microscopy > with $1550 CMOS cameras) ... > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_ar > ticles_s41598-2D018-2D19981-2Dz&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc > 04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrm > PqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=Uu8NR6naGZycBiUtnau3E1wR4y4fN > ok6ZujZHMvXGWY&e= > > Moffitt et al 2018 ... > https://urldefense.proofpoint.com/v2/url?u=http-3A__science.sciencemag > .org_content_362_6416_eaau5324.long&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeE > lZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=Q > hUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=J2hElzRpmEhoCdpN3AeONt9_O > 6naYQrBAiIw8h7zE5c&e= and commentary > https://urldefense.proofpoint.com/v2/url?u=http-3A__science.sciencemag > .org_content_362_6416_749&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3E > xJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07F > T6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=9lrtHfkyrZ91YwO1mX9XIgo7iM-qm3zflvT > o_y1TYWk&e= > > *** > > Some resolution numbers: > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * > wqavelength / NA (I routinely drop the 0.61 from 0.61) > > 0.6 * 500 / 1.4 = 214 nm > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 nm (if > pixel size matched or interpolate optimally). > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 > Principles > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > resolution wrt 1 and smaller pinhole size, source of the values below, > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons > throughput (which doesn't matter if target is photostable). > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan (and > FastAiryScan). > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons throughput. > > Most modern point scanning confocal microscopes have a 405 nm laser, > so if using BV421 (and ignoring potential photobleaching for a > moment), > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > or in reflection mode, i.e. nanodiamond or nanogold, > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection implies no > photobleaching, so infinite number of photons (though also no > blinking, so not usually eligible for precision localization) ... > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > and not going completely exotic with NA (i.e. 1.65), if perfectly > refractive index match with a fairly conventional 1.49 NA lens, and > inreflectance: > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > I think I'd rather invest a DNA-PAINT friendly rig than deal with 157 > to > 101 nm. > > DNA-PAINT makes resolution irrelevant, if you use it (and don't run > out of disk space or time or money), since precision localization is > resolution divided by square root of number of photons, ex. 250 nm XY > resolution / sqrt(1,000,000) = 0.25 nm, and could increase number of > photons per target further, but why bother? > > *** > > point scanning confocal microscopes are also great platforms for > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much faster than > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih. > gov_pubmed_18387308&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII > ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_ > r1_9otpqB337-xfUGpGNb7ONvc&s=2wsUl3TkcW6hCeGZ7XS0HuDA1fe27y-YJTFdjfrfS > 2g&e= > > *** > > p.p.s. Disclosures I am ... > > 1. currently hosting a Leica THUNDER Imager tour event (ends Monday > 3/18/2019 afternoon) ... see pdf download page, > > THUNDER Technology Note > THUNDER Imagers: How Do They Really Work? > > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.leica-2Dmicro > systems.com_science-2Dlab_thunder-2Dtechnology-2Dnote&d=DwIFaQ&c=j5oPp > O0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV > 7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=xRDjJDP > NeqFeYwha2XGXbIpLiWeX8IbVAzG-lzoxIRo&e= > > 2. hosting Nikon confocal demos in May 2019. > > 3. aiming to co-host with ISS a FastFLIM (one day) mini-symposium this > summer. > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > https://urldefense.proofpoint.com/v2/url?u=https-3A__www.ncbi.nlm.nih. > gov_pubmed_28261321&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeII > ZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_ > r1_9otpqB337-xfUGpGNb7ONvc&s=8YR5g4lC-nPSNY3Qrraw1xlFP0fPAaHtqKsOQbRHr > NQ&e= > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cg > > i-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48Dtse > > deElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5b > > U&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGpGNb7ONvc&s=3LqjIRKGooGbWufKQI > > fCOyBhknMhzM4msy9LD6YtGwo&e= Post images on > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d= > > DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDi > > PlC50tp7rW2nBkvV7fujQf0RknE5bU&m=QhUrmPqL07FT6Fex_r1_9otpqB337-xfUGp > > GNb7ONvc&s=LwlU9Ttd8-NvvVIj2xG11Fkz5KupvCoj80DexAghEHY&e= and > > include the link in your > posting. > > ***** > > > > Hi. > > There are software solutions that are able to create image data from > wide-field and even microscope systems with seemingly similar quality > to that obtained from confocal systems. > > > > The comparison of acquisition vs. software is essentially a > > comparison > of image acquisition vs. image processing. While a software solution > is way cheaper than a hardware solution if it is able to produce image > data with equal quality why would anyone choose to invest to a confocal anymore? > > > > I’m looking forward to a vidid discussion! > > > >> m > ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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Tim Feinstein |
Re: General question: Software vs. hardware
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Csúcs Gábor-3 |
Re: General question: Software vs. hardware
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Connected to the same thread I would like your opinion about the new Leica davalopment (they call it "computational clearing" and sell it under the name "Thunder". They are very resistant to tell any mathematical details about it (even in a recently published white paper). Based on some rudimentaqry explanation it seems (really my subjective interpretation)that they do a wavelet conversion, filter out the large spatial components (claiming that those correspond to the out-of-focus background) and then transfer back the data. With this they claim to have the same effect as structured illumination (e.g. ApoTome) or a spinning disk (though this is a clear overstatement). Any thoughts from the community? Greetings Gabor |
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Craig Brideau |
Re: General question: Software vs. hardware
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** If you don't know how it works, you can't use it. There's always a chance the features of your sample will fall on some edge case of the mystery algorithm and give you BS results. If you can't prove the algorithm works correctly on your samples it's not solid data, and if you don't know the algorithm used then you can't do that. Craig On Mon, Mar 18, 2019 at 2:04 PM Csúcs Gábor <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Connected to the same thread I would like your opinion about the new Leica > davalopment (they call it "computational clearing" and sell it under the > name "Thunder". > They are very resistant to tell any mathematical details about it (even in > a recently published white paper). Based on some rudimentaqry explanation > it seems (really my subjective interpretation)that they do a wavelet > conversion, filter out the large spatial components (claiming that those > correspond to the out-of-focus background) and then transfer back the data. > With this they claim to have the same effect as structured illumination > (e.g. ApoTome) or a spinning disk (though this is a clear overstatement). > Any thoughts from the community? > > Greetings Gabor > |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I have never seen anyone do that, but a reasonable way to test whether improvement by deconvolution is real would be to apply it to a low-resolution image and compare to unprocessed high-resolution image ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Craig Brideau <[hidden email]> Sent: Monday, March 18, 2019 4:16 PM To: [hidden email] Subject: Re: General question: Software vs. hardware ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** If you don't know how it works, you can't use it. There's always a chance the features of your sample will fall on some edge case of the mystery algorithm and give you BS results. If you can't prove the algorithm works correctly on your samples it's not solid data, and if you don't know the algorithm used then you can't do that. Craig On Mon, Mar 18, 2019 at 2:04 PM Csúcs Gábor <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Connected to the same thread I would like your opinion about the new Leica > davalopment (they call it "computational clearing" and sell it under the > name "Thunder". > They are very resistant to tell any mathematical details about it (even in > a recently published white paper). Based on some rudimentaqry explanation > it seems (really my subjective interpretation)that they do a wavelet > conversion, filter out the large spatial components (claiming that those > correspond to the out-of-focus background) and then transfer back the data. > With this they claim to have the same effect as structured illumination > (e.g. ApoTome) or a spinning disk (though this is a clear overstatement). > Any thoughts from the community? > > Greetings Gabor > |
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Benjamin Smith |
Re: General question: Software vs. hardware
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In reply to this post by Tim Feinstein
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Not sure if this has been said, but this is basically like asking "screwdriver vs. wrench". A laser scanning confocal microscope is not superior to a compound microscope, and the converse is also true, they are different tools for different tasks. This is one of the key points I try to get across to students when I teach them about microscopy, we have macro, spinning disk, 2P, light sheet, stereo, confocal, STED, STORM, TEM, SEM, AFM, FIB-SEM, etc., etc, for a reason. They all excel at tasks that other systems struggle with. Along these lines, here are two scenarios: Scenario 1) You want to get a kHz sample rate of a voltage dye in a cultured neuron. In this case, a compound microscope with deconvolution (or likely even just a simple high-pass filter) is the clear winner as all the pixels in the frame are temporally correlated (as long as you have CCD or global shutter CMOS), and the frame rate will be much higher than with confocal, even with the most cutting edge technologies. Scenario 2) You want to measure the volume of densely packed nuclei using DAPI in a whole-mount sample. Deconvolutions will quickly fall apart on this task simply because the deeper you go, the vast majority of the total signal is from out of focus light (much like trying to image a faint star right next to the sun). This means that the amount of information you have about the sample plane itself becomes nearly non-existent. Conversely, since confocal microscopes perform the deconvolution before light gets to the detector, you more or less eliminate this bottleneck caused by the dynamic range of the detector. Also, one quick point about deconvolutions. Unless you measured the PSF in the sample (such as using TetraSpeck beads) at a higher resolution than you acquired your image, you are not adding any information about the sample. Rather, you are whittling away information you wish to discard (i.e. it is a lossy process, much like JPEG compression). Along these lines, iterative blind deconvolutions are allowing a computer to guess what information should be removed. Thus, just because the image looks better does not necessarily mean it is correct, otherwise STED, STORM, AFM, and cryo-EM would be obsolete. Just my own two cents, Ben Smith On Mon, Mar 18, 2019 at 11:52 AM Feinstein, Timothy N <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Hi Alison, > > The signal to noise question strikes me as the key question WRT decon. > Most software packages default to a setting that seems unrealistic for > confocal imaging (ie SNR=20), and this leads to a lot of patterning > artifacts in deconvolved images. At such high SNR any pixel of data is > treated as 'real', so background noise gets processed into weird moire-type > patterns. This also happens when the algorithm doesn't quite converge and > you let it run for too many iterations (the default iteration #s tend to be > set high as well). It's critical then to tell the software (when possible) > that your SNR is low, so it gets much more aggressive about detecting and > removing random noise (at the expense of losing details in the 1-3 pixel > range). I would recommend folks take care if using any package that > doesn't let you correct the SNR. > > Honestly, when SNR is set properly I find that noisy images benefit a lot > more from decon than high-quality cover candidate-type pics. In addition > to sharpening and Z blur removal the noise and background removal becomes > more dramatic (and beneficial for quantitation) the more background and > noise there is. This comes up a lot when live imaging when we use resonant > scanning for speed and low photodamage, but have higher noise as a > trade-off. I'd say that as long as you can image at 1.5x-2x Nyquist > resolution (and the extra time/expense is worth it), even super-noisy > images will benefit quite a bit. > > I understand that we're crossing fingers for the day when everyone has > GPU-enabled decon running seamlessly during acquisition. For that to work > the algorighms need to automatically (accurately) estimate SNR. That > doesn't seem like an insurmountable challenge, but I haven't seen it yet. > > > Best, > > > TF > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > > > > On 3/18/19, 11:07 AM, "Confocal Microscopy List on behalf of Alison > North" <[hidden email] on behalf of > [hidden email]> wrote: > > > Hi all, > > So this is a very timely discussion because I have been discussing > with > my staff whether there are data sets that should NOT be deconvolved, > and > if so, how does one decide that? I too attended Jim Pawley's > wonderful > course (he was certainly a huge character as well as an incredibly > fantastic microscopist, and he will certainly be remembered by all!), > so > I have generally worked under the assumption that one should > deconvolve > all confocal data. But I am also very aware of the potential for > artifacts if a data set isn't "good enough" for deconvolution. > Obviously the ideal situation is to acquire an optimal data set - > well-prepared sample, bright staining, Nyquist sampling etc. etc., and > a > high S:N ratio - and by sticking to these rules, our deconvolved > DeltaVision images or confocal images of fixed samples have always > looked great. But nowadays we are faced with different scenarios, > particularly when you are attempting to do very rapid imaging of live, > weakly expressing cells, while attempting to minimize phototoxicity. > In > that case you can end up with pretty lousy S:N ratios, because > maintaining cell viability or imaging fast enough is more critical. > For example, I have a lovely new iSIM in my lab, for which the initial > resolution increase is achieved by the hardware, but the second step > in > resolution increase is via deconvolution. The whole point of this > instrument is for rapid, super-resolution imaging - so we can't simply > increase exposure times to improve S:N, turning up the laser power > will > obviously kill the cells, and we can't increase the pixel size or > we'll > lose the resolution. And I assume a lot of the new types of > super-resolution instrument out there must leave you facing the same > issue, since live cell imaging invariably forces you to compromise > somewhere within the imaging triangle (or hexagon, or whatever we've > got > up to now!). > > Therefore my question is, are there papers out there which have > compared > deconvolution algorithms and looked at the potential for artifacts on > really low S:N images, which we could use to advise our researchers on > what is the minimum you can get away with before you really shouldn't > be > deconvolving the data set at all? Also, are there papers showing the > effect of undersampling in the z-axis on the resulting deconvolved > images (as is often the case on our spinning disk system)? I haven't > managed to find any yet (though I confess I've been too busy with > other > stuff to spend too many hours searching!), so if anybody could point > me > to some good references I'd be most grateful. I have spoken with > several renowned microscopists about whether deconvolution is always a > good idea under such circumstances, and the gut reaction appears to be > no, but I could do with some hard and fast validation for teaching > purposes. > > Many thanks in advance! > > Alison > > > > On 3/18/2019 9:56 AM, Feinstein, Timothy N wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DvfjahhKueherhfphxmr-Smw_FhB4QUlg-FsBATM1kl0%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=7V6L2MVDHIM%2BwC0pAgCi3pgsBuFzbd0XQQWFWD%2BTJfY%3D&reserved=0= > > Post images on > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttp-3A__www.imgur.com%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DedOjY0NZEIhVq-zWzgNHHDaTrutHrPj1Rl6rcwE_B4M%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=1c1u00xUm3GBfY%2BbuasN%2FzjLdqR5THU38W7oITOW9k0%3D&reserved=0= > and include the link in your posting. > > ***** > > > > Avi, I really agree with your point. I feel that people to > deconvolve any time spatial information is critical, whether they're using > widefield, CLSM, spinning disc, or light sheet. It's true that > deconvolving adds time and data volume and especially cost, but in trade > you get an image that is substantially sharper, with reduced noise and > background, and more quantitatively accurate*. > > > > Regarding whether to just go with a point scanning confocal, I don't > see it as a simple question of better or worse**. A nuclear-cytoplasmic > translocation assay with monolayer cells works just as well on a widefield, > and (in my experience!) many types of biosensor assay work better with a > properly set up widefield. The 16-bit depth of widefield images is nice > for quantitation, and modern sCMOS cameras have by far the best acquisition > speeds. I don't know whether widefields still have a more linear > relationship between sample brightness and detected signal, but the last > time I checked that was still true. > > > > (*) Deconvolution is quantitatively useful as long as people make > sure to tell the software to preserve the original intensity values. One > of my complaints about Hyvolution was that you could not do that, so I just > used the Huygens package that came with it. I don't know whether Lightning > gives you that option...if not then caveat emptor. > > > > (**) My advice mostly applies to turnkey stuff that any lab can > implement, not exotic techniques available to folks with specialists or > engineers on hand. > > > > Best, > > > > > > T > > Timothy Feinstein, Ph.D. esearch Scientist > > Department of Developmental Biology > > University of Pittsburgh > > > > > > On 3/18/19, 5:07 AM, "Confocal Microscopy List on behalf of Avi > Jacob" <[hidden email] on behalf of [hidden email]> > wrote: > > > > > > I'll point out, that you can, of course, deconvolve confocal > images too. > > So, while you can indeed get near confocal quality with > well-acquired wf > > data after deconvolution, you can also get near SR quality when > > deconvolving a well-acquired stack from a confocal. And then > you can also > > deconvolve a SR stack and get... well you get the idea! It's > like an arms > > race. > > I have the Hyvolution and had access for a couple of weeks to > the Lighting, > > and now confocal images look blurry to me. > > Avi > > > > -- > > Avi Jacob, Ph.D. > > Kanbar Light Microscopy Unit > > The Mina & Everard Goodman Faculty of Life Sciences > > Bar-Ilan University, Ramat-Gan 529002, Israel > > > > > > > > On Sun, Mar 17, 2019 at 6:04 PM George McNamara < > [hidden email]> > > wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go > to: > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DefIAtI3pbBSoyhJucB0DkDu4RXkFkgBZfGrO4PiXrfc-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D9Uk5ZZzc8LP7V7Oj2aFSRbVluY0mpE-7XPLYdsyviMk%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=Jv2%2FvAnr1srE%2BjcBXMWe2mVTvO9rowobKYY65DWIRTo%3D&reserved=0= > > > Post images on > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DMNM8i3iDaQuMA9LF766-252FyvCyp94jmie4IaC5qIEWclA-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DGy99yAWcbr6KHUA0kYKb4K-wOHL4iNGJsDecMfZ8X-M%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=2N%2BmlW5RE%2BAFuG%2FuM68FeI9LOfI1m2PlNu23S9eIQts%3D&reserved=0= > and include the link in your posting. > > > ***** > > > > > > Hi Mika, > > > > > > White et al 1987 ( > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fjcb.rupress.org-252Fcontent-252F105-252F1-252F41.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Da6oZCfeD7Gt4nYMS89PcklnveCdDLLkksafp3tCyld0-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D67LyqPF2rBiBDSWJVZeG7TpJz1o4MRDf8ZyLysbeKx4%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=R3V0RHiTDpGkJUJyI8Q7SDJZjKdk3uDMgmBEPLrzcUw%3D&reserved=0= > ) made a > > > compelling case for point scanning confocal microscopes: > collect just > > > the in focus light with instant gratification. The case has > not changed, > > > the hardware (especially data deluge side) has gotten a lot > better. I > > > note that both widefield detectors and PMT/APD/hybrid > detectors/others > > > have gotten a lot better in the 32 years from 1987! As have > the optics > > > and automation. > > > > > > Paul Goodwin 2014 ( > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F24974028-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DGDve8BuxhLDAdBPKywRQoMTYV-252BqSk9aMcz2WrZCHPU8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DPZ10eaR5B-jwZmf5y920Z41iJYUJcji_VsufABIeX84%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=dIZN7CiQOTIt24N0mwi0QgDFkcFcVScVNxaoDuS24HM%3D&reserved=0= > ) made > > > a nice case for quantitative deconvolution microscopy (and on > very high > > > quality specimens, ~10% improvement in resolution compared to > simple > > > widefield), but historically slow. > > > > > > Now, with 'instant gratification' spatial deconvolution, > thanks to the > > > GPU revolution (NVidia RTX Titan~16 Teraflops [S.P.], 24 Gb > ram, $2500 > > > ... not including the deconvolution software module price), > widefield, > > > spinning disk (and slightly exotic variants like iSIM, DMD > based, etc, > > > see also new THUNDER Imagers [see p.p.s.]), multiphoton (I'm > excited > > > about the price point of recent fiber lasers, which could > become much > > > better price if achieve 'economy of scale'), and of course, > point > > > scanning confocal microscopes. > > > > > > Spatial deconvolution (especially if someone $uccessfully > implements > > > joint spatial deconvolution and spectral unmixing, multiple > cameras - > > > for 4 cameras see Babcock 2018, mentions aiming for 8 > cameras) helps > > > with Expansion Microscopy and/or DNA-PAINT, to go > super-resolution ... > > > really single molecule counting (and DNA-PAINT eliminates the > classic > > > issue of PALM/STORM/FPALM of not counting every molecule). > Sure, > > > DNA-PAINT (like STORM etc) have the issue of a whole lotta > images > > > acquired. Data deluge: who cares? Jerome & Price's 10th > commandment of > > > confocal imaging is: "10. Storage Media Is Essentially Free > and Infinite". > > > > > > More significantly, DNA-PAINT and related methods (single > molecule RNA > > > FISH, scRNAseq -> MERFISH = Moffitt 2018 as example, etc) > also enable > > > multiplex -- with single molecule counting -- to whatever > plex is needed > > > to answer the 'biological question(s)' being posed. > > > > > > All that said, the installed base of research grade point > scanning > > > confocal microscopes is large (5000+) and efficient at > acquiring high > > > quality images, to the point that user's sample preparation > (and > > > avoidance of purchasing stuff from 'Santa Crap' and similar > companies) > > > is much more limiting than the microscopes. > > > > > > George > > > > > > p.s. a couple of references not included in above: > > > > > > W. Gray (Jay) Jerome, Robert L. Price 2018... Basic Confocal > Microscopy > > > second edition > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Flink.springer.com-252Fbook-252F10.1007-25252F978-2D3-2D319-2D97454-2D5-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3D-252FMQlH4t81Pk9tNTHR8fsn1tQc0JAc2nc-252BnO2gdk60Us-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D1JXaiNNf-bzSdjywfRJ8gowB9Yq0uGNulQMlGeSMxkE%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=3ua69ygadJRYIJltAT7qPoJzNd%2B16PWCCeNLQF13WIg%3D&reserved=0= > > > > > > Expansion ... X10 protocol ... Truckenbrodt 2019 Nat Protoc, > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41596-2D018-2D0117-2D3-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DCF-252Fp9frimbrxZiMDwLWZjzuyoyQbCuKp4EcvaL3Wmmw-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYeXbqWFX0mMxO3nVRt59pt6RJYlwIPBMZRePQbiF1yU%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288894390&sdata=TEBfgj6Gg%2Bt02BMyFZCLb8%2Feb5nOyAJmnftouvPd1qY%3D&reserved=0= > > > > > > DNA-PAINT acronym soup review ... Nieves 2018 Genes, > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.mdpi.com-252F2073-2D4425-252F9-252F12-252F621-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3Diwx3Z1Pgr4a2SObO9F47jEtqlfsYQFk2R4gu0Qv1uJM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DtJY5yunlGClYXKHrXuCDe5KU8D-qhc1ZS26_KuNdXpE%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=RUgIPB%2Fi0n6i28lOhKZAGSrlyirYALd0l75r5qhZCMk%3D&reserved=0= > > > > > > Babcock 2018 (4 --> 8 cameras, single molecule localization > microscopy > > > with $1550 CMOS cameras) ... > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.nature.com-252Farticles-252Fs41598-2D018-2D19981-2Dz-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257860747-26amp-3Bsdata-3DwN-252FHP30wNUE0-252BJWeFJIJFsBk32ZhD4DfKi9gc3ZTz2c-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DTIL4NaeETsqkjycmtWw_CnoEWvrpY1Tkr_SAmz-QkRM%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=D26AicKRsRoIaCGZdaxaF5OKXfGuo8yS0zvSVgmoBwY%3D&reserved=0= > > > > > > Moffitt et al 2018 ... > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252Feaau5324.long-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DFR98V6ldS9Q38wI59kly9U8pCzp92Vzc1J6T8ydCU9w-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3D55vzrzqpm3n5Z7logfovDr0N73h1gF7gldtYUSkZ6c0%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=CQMKMRjzbJVLgZjtpCzzZSwaNvCbt72baFvqppyebSI%3D&reserved=0= > and > > > commentary > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fscience.sciencemag.org-252Fcontent-252F362-252F6416-252F749-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3Do0B379uuh-252FrB6gS9n6lG-252BjAZeztiYHZVmICwX4ghKh8-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DXeBps6CWTMM197evmy1n1uC5qOcvU8mAN95bEPmieiQ%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=um%2F%2FPNffQLOyrTsQcJymSuR2XwRcVnNMwMgQ6qYy19A%3D&reserved=0= > > > > > > *** > > > > > > Some resolution numbers: > > > > > > 1.4 NA objective lens ... 500 nm wavelength .. dxy = 0.61 * > wqavelength > > > / NA (I routinely drop the 0.61 from 0.61) > > > > > > 0.6 * 500 / 1.4 = 214 nm > > > > > > widefiel deconvolution (re: Goodwin 2014) ~10% better ... 193 > nm (if > > > pixel size matched or interpolate optimally). > > > > > > point scanning confocal -- Zeiss has a nice PDF, "Zeiss 2008 > Principles > > > - Confocal Laser Scanning Microscopy" (see fig 10) on confocal > > > resolution wrt 1 and smaller pinhole size, source of the > values below, > > > > > > 1 Airy unit: 0.51 * 500 / 1.4 = 182 nm. > > > > > > 0.5 Airy unit ... 0.44 * 500 / 1.4 = 157 nm ... ~0.25 photons > throughput > > > (which doesn't matter if target is photostable). > > > > > > 0.2 Airy unit ... you can ask your Zeiss rep about AiryScan > (and > > > FastAiryScan). > > > > > > 0.1 Airy unit ... 0.37 * 500 / 1.4 = 132 nm ... ~0.10 photons > throughput. > > > > > > Most modern point scanning confocal microscopes have a 405 nm > laser, so > > > if using BV421 (and ignoring potential photobleaching for a > moment), > > > > > > 1 Airy unit: 0.51 * 421 / 1.4 = 153 nm. > > > > > > or in reflection mode, i.e. nanodiamond or nanogold, > > > > > > 1 Airy unit: 0.51 * 405 / 1.4 = 147 nm ... and reflection > implies no > > > photobleaching, so infinite number of photons (though also no > blinking, > > > so not usually eligible for precision localization) ... > > > > > > 0.1 Airy unit: 0.37 * 405 / 1.4 = 107 nm > > > > > > and not going completely exotic with NA (i.e. 1.65), if > perfectly > > > refractive index match with a fairly conventional 1.49 NA > lens, and > > > inreflectance: > > > > > > 0.1 Airy unit: 0.37 * 405 / 1.49 = 100.57 nm > > > > > > I think I'd rather invest a DNA-PAINT friendly rig than deal > with 157 to > > > 101 nm. > > > > > > DNA-PAINT makes resolution irrelevant, if you use it (and > don't run out > > > of disk space or time or money), since precision localization > is > > > resolution divided by square root of number of photons, ex. > 250 nm XY > > > resolution / sqrt(1,000,000) = 0.25 nm, and could increase > number of > > > photons per target further, but why bother? > > > > > > *** > > > > > > point scanning confocal microscopes are also great platforms > for > > > F-Techniques, such as FastFLIM (aka rapidFLIM, etc, much > faster than > > > classic TCSPC slow FLIM), FCS, FCCS, see Liu 2008, > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F18387308-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DZQTosrSoPFpCa8Y3IdEa9Xz-252B4RHC8JO4gBppHmzkazo-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DmeRUIZdF3FLQZGyok4cxyc4AP4yq2lXX41Vt8q9WMmw%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=XTEH306i5ez7vKq2cHKCVQlSphNiFIdnqIXmwnOmyNw%3D&reserved=0= > > > > > > *** > > > > > > p.p.s. Disclosures I am ... > > > > > > 1. currently hosting a Leica THUNDER Imager tour event (ends > Monday > > > 3/18/2019 afternoon) ... see pdf download page, > > > > > > THUNDER Technology Note > > > THUNDER Imagers: How Do They Really Work? > > > > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.leica-2Dmicrosystems.com-252Fscience-2Dlab-252Fthunder-2Dtechnology-2Dnote-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DauRUORuEKAir87-252Bbw7RHyTR9IxxrLYl1g3bFBiRTXBM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DLmFGYl_Icu6b9AAIYnHkoiH26bRZ688ODEc9I6k8yco%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=5ZGLmCZt85tKxH1CJYM%2Bvul5S4qXw9%2FOnyWG80m2QxM%3D&reserved=0= > > > > > > 2. hosting Nikon confocal demos in May 2019. > > > > > > 3. aiming to co-host with ISS a FastFLIM (one day) > mini-symposium this > > > summer. > > > > > > 4. an unpaid advisor for Gary Brooker for FINCH/CINCH, re: > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttps-253A-252F-252Fwww.ncbi.nlm.nih.gov-252Fpubmed-252F28261321-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DuVw0kiW1baZV0RJv-252Bk1ObKznhw51pemnCMehjHkUa2g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DKC1HMkiDzpE2DLsuj1I2nHvopwFVMMc0GVecDV-fWkM%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=QC0LyEp9hbSKOgpV%2Fi%2B%2FABGDX4az02fRVdMZ4Ovufps%3D&reserved=0= > > > > > > > > > On 3/17/2019 10:43 AM, Mika Ruonala wrote: > > > > ***** > > > > To join, leave or search the confocal microscopy listserv, > go to: > > > > > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DdHOCjeBda4sSZPf-252FB2-252B5Sv3Q8Qzcs708p4we8vHf-252FIg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DqZ-tdRwhHG172zv4uHPeAJMNeIIHNxozhQudV-hF2kE%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288904398&sdata=BnCAxorVJgPSFw70Kr%2B401vq4hMlZhvzdkivum9XGNE%3D&reserved=0= > > > > Post images on > https://nam05.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__nam05.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Ctnf8-2540PITT.EDU-257Ce2895884dda94858a22408d6ab8114ff-257C9ef9f489e0a04eeb87cc3a526112fd0d-257C1-257C0-257C636884968257870756-26amp-3Bsdata-3DcnhzoWOP4RwDR622fn447aQVxW8ZBI3D0utze67RiGg-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3DJeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg%26r%3DRBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo%26m%3DjqVgS0XjNSmttSUahDdaDaAsBeK67RlyWHCj8N7ZMJw%26s%3DYzF36y8eV4SG3E0LJEDu-9AMm3Nh05dwp_XKhB6BUyU%26e&data=02%7C01%7Ctnf8%40PITT.EDU%7Cdc51ccda0c794046d93408d6abb362a8%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636885184288914407&sdata=ec0HIKuOMVqlLU7knyQ1JdS1B6c1BMCIb%2FYsHIFdYbM%3D&reserved=0= > and include the link in your > > > posting. > > > > ***** > > > > > > > > Hi. > > > > There are software solutions that are able to create image > data from > > > wide-field and even microscope systems with seemingly similar > quality to > > > that obtained from confocal systems. > > > > > > > > The comparison of acquisition vs. software is essentially a > comparison > > > of image acquisition vs. image processing. While a software > solution is way > > > cheaper than a hardware solution if it is able to produce > image data with > > > equal quality why would anyone choose to invest to a confocal > anymore? > > > > > > > > I’m looking forward to a vidid discussion! > > > > > > > >> m > > > > > > > > -- > Alison J. North, Ph.D., > Research Associate Professor and > Senior 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 > > > -- Benjamin E. Smith, Ph. D. Imaging Specialist, Vision Science University of California, Berkeley 195 Life Sciences Addition Berkeley, CA 94720-3200 Tel (510) 642-9712 Fax (510) 643-6791 e-mail: [hidden email] http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/> |
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