confocal detectors and deconvolution
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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 colleagues, we are looking for a new confocal microscope, and I have two questions so far. 1. Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then. 2. Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra. Thank you Mike Model Kent State University |
 
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Vitaly Boyko-2 |
Re: confocal detectors and deconvolution
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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 Mike, there is no big difference between HyDs and GaAsP detectors. Leica SMD HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more for the GaAsP.I am not aware of linear deconvolution algorithms for confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and not suitable for accurate intensity based image analysis.Recent integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new era to biology letting biologists quantify their data and image biological molecules at their physiological concentrations. However, most of biologists seems to be "scared" of basic equations...If you are located in the US, Leica provides much better Service and Application support compared to Zeiss. I do not have much experience with Olympus and Nikon on the confocal side. If you have any specific questions, please let me know. VitalyMCCF/MSKCC On Thursday, May 10, 2018, 10:01:36 AM EDT, MODEL, MICHAEL <[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. ***** Dear colleagues, we are looking for a new confocal microscope, and I have two questions so far. 1. Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then. 2. Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra. Thank you Mike Model Kent State University |
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George McNamara |
Re: confocal detectors and deconvolution
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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 Mike, My comments are in addition to Vitaly's. * EVERY confocal microscope and every widefield microscope should have GPU deconvolution immediately downstream, routinely, with 'instant gratification'. Both Leica HyVolution2 (with SVI Huygens under the hood, mostly seamless) and new Olympus are checkbox or dropdown to turn on deconvolution (Olympus GPU deconvolution just launched). * Any Deconvolution artifacts are present in the raw data, just not so obvious. Fluorescent beads of very high R.I. under water (R.I. 1.33) or air (1.0) produce excellent artifacts. If you write an NIH grant specifically to study these artifacts, don't expect to get funding. Near perfect R.I. matching is now readily doable for both live cells (https://www.ncbi.nlm.nih.gov/pubmed/?term=boothe+iodixanol ... if you have a Silicone oil objective lens) or fixed cells (R.I. ~1.518 if need oil immersion using thermoFisher/Mol Probes Prolong Glass, can also check out Marker Gene's Opti-Bryt, and check EMSdiasum's web site for other mounting media). * We just ordered our Olympus FV3000RS on IX83 stand, several bullet points related to this purchase: -- thanks to NIH 'confocal' study section and U.S. taxpayers for approving our S10 shared instrumentation grant proposal. -- We are getting four GaAsP detectors inside, plus two external GaAs "NIR" PMTs (see Jim Pawley's handbook chp 2 fig 2.10 for QE curves: GaAs is superior in >700 nm, though I don't know the entire electronics chain ... and yes, I wish Olympus sold 90% Q.E. APDs for this, and APDs could have fit in budget, we'll be ok). Seven laser lines, including 730 nm for the NIR stuff. - RS = resonant scanner, yes, standard high res and RS fit in budget. Ever since Jonathan Boyd (who moved from Leica to Medimmune) showed me RS is superior when matching pixel dwell time (8000 Hx * 100 scans vs 800 Hz * 10 scans vs 80 Hz one scan), likely due to less photobleaching, RS with a little averaging (or summing for HyD photon counters) is the way to go, if field of view, zoom, ok. - The Olympus software released in mid-April (2018) now is able to use NVidia GPU. Negotiate with your salespeople (i.e. Leica [SVI Huygens for HyVolution2, don't know if they also do their own thing for FALCON), Olympus, Nikon may also be GPU, Microvolution, AutoQuant) on price of the deconvolution AND what GPU card(s) is(are) going into the workstation (might end up being sold a cheap GPU and then going out and buying the right one ... make sure the case and power supply can handle it/them). Also important to know if the workstation can handle say 3 Quadro GV100's (three double width PCIe cards) or say 4 V100's on the motherboard, see for example, https://www.supermicro.com/products/system/4u/7048/sys-7048gr-tr.cfm (sure, one FV100 or one V100 is $9000, but one or more would enable 'instant gratification' on a possibly $500K confocal or $900K light sheet rig ... I also note that 100Gbe Ethernet cards are $800 so if your new PC quote is still 1gig, tell the salesperson to fix it ... sure, also need more cards, and probably a switch and 100Gbe NAS to send the data to). - Olympus "SSU" ultrasonic stage is very nice. If you go with Olympus, get it (or at least demo it and decide for yourself). You can ask other companies if they have the same thing. * I manage a Leica SP8 confocal microscope with 2 HyD detectors and HyVolution2, works great (I see I have more edits), http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope * looks like Nikon's spectral confocal unmixes in by fluorophore (finally a big confocal company gets it right), whereas Leica (LAS X on SP8, same as earlier LAS on SP5) and ZEN Black (which might or not be improved in 'zen one') unmix brightest channel first (bad idea if ever try to do controls, especially "fluorescence minus one" controls). * Geoff Daniels (Leica confocal sales) explained FALCON ... very impressive sounding. If you have the money, four FALCONhyD's should be great. If you have only money for one or two of them, start with that (the pulsed white light laser likely costs more than four HyDs). Ideally get an X1 port to enable more detectors (i.e. 2 or more APDs for NIR). * Zeiss LSM880 with Airyscan (I'm not sure why anyone would buy an 880 without Airyscan ... seems like a 780 and marketing added 100). * There are a lot of very interesting "other scopes" out there, mention three: - Abberior Instruments - see http://www.abberior-instruments.com/site/assets/files/1057/abberior_instruments_adaptive_optics_easy3d_sted_deep_imaging.png - ISS Alba and Alba-STED. - confocal.nl "rescan". - whatever AO LLS thingy Eric Betzig recently published ... hopefully whatever company eventually distributes it does not botch the product rollout. Note quite comprehensive table of microscope vendors is one of the tables in my (open access) Unit https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cphg.42 which is slightly newer than my online table http://www.geomcnamara.com/light-microscopy-websites enjoy, George p.s. if making "channel unmixing" matrix, I think photon counting is the way to go (HyD's), and do a lot of line and frame accumulation to get the best data for computing the coefficients (from single stained controls). For example, 16 line * 16 frame accumulations = 256 (the current max in leica LAS X), which produces bigger numbers than my experimental scanning 'standard of care' of 10 line accumulation (no frame accum). Sure, I am assuming no photobleaching (If bleaching was a concern, I would repeat, compare, and if no bleaching, add the two). On 5/10/2018 11:19 AM, Vitaly Boyko 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 Mike, > there is no big difference between HyDs and GaAsP detectors. Leica SMD HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more for the GaAsP.I am not aware of linear deconvolution algorithms for confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and not suitable for accurate intensity based image analysis.Recent integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new era to biology letting biologists quantify their data and image biological molecules at their physiological concentrations. However, most of biologists seems to be "scared" of basic equations...If you are located in the US, Leica provides much better Service and Application support compared to Zeiss. I do not have much experience with Olympus and Nikon on the confocal side. > If you have any specific questions, please let me know. > VitalyMCCF/MSKCC > On Thursday, May 10, 2018, 10:01:36 AM EDT, MODEL, MICHAEL <[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. > ***** > > Dear colleagues, > > > > we are looking for a new confocal microscope, and I have two questions so far. > > 1. Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then. > > 2. Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra. > > > > Thank you > > > > Mike Model > > Kent State University -- George McNamara, PhD Baltimore, MD 21231 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome) http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 http://confocal.jhu.edu July 2017 Current Protocols article, open access: UNIT 4.4 Microscopy and Image Analysis http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract supporting materials direct link is http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 figures at http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures |
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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. ***** Thank you, George, Owen, Vitaly and Thomas! Mike -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of George McNamara Sent: Thursday, May 10, 2018 10:46 PM To: [hidden email] Subject: Re: confocal detectors and deconvolution ***** 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 Mike, My comments are in addition to Vitaly's. * EVERY confocal microscope and every widefield microscope should have GPU deconvolution immediately downstream, routinely, with 'instant gratification'. Both Leica HyVolution2 (with SVI Huygens under the hood, mostly seamless) and new Olympus are checkbox or dropdown to turn on deconvolution (Olympus GPU deconvolution just launched). * Any Deconvolution artifacts are present in the raw data, just not so obvious. Fluorescent beads of very high R.I. under water (R.I. 1.33) or air (1.0) produce excellent artifacts. If you write an NIH grant specifically to study these artifacts, don't expect to get funding. Near perfect R.I. matching is now readily doable for both live cells (https://www.ncbi.nlm.nih.gov/pubmed/?term=boothe+iodixanol ... if you have a Silicone oil objective lens) or fixed cells (R.I. ~1.518 if need oil immersion using thermoFisher/Mol Probes Prolong Glass, can also check out Marker Gene's Opti-Bryt, and check EMSdiasum's web site for other mounting media). * We just ordered our Olympus FV3000RS on IX83 stand, several bullet points related to this purchase: -- thanks to NIH 'confocal' study section and U.S. taxpayers for approving our S10 shared instrumentation grant proposal. -- We are getting four GaAsP detectors inside, plus two external GaAs "NIR" PMTs (see Jim Pawley's handbook chp 2 fig 2.10 for QE curves: GaAs is superior in >700 nm, though I don't know the entire electronics chain ... and yes, I wish Olympus sold 90% Q.E. APDs for this, and APDs could have fit in budget, we'll be ok). Seven laser lines, including 730 nm for the NIR stuff. - RS = resonant scanner, yes, standard high res and RS fit in budget. Ever since Jonathan Boyd (who moved from Leica to Medimmune) showed me RS is superior when matching pixel dwell time (8000 Hx * 100 scans vs 800 Hz * 10 scans vs 80 Hz one scan), likely due to less photobleaching, RS with a little averaging (or summing for HyD photon counters) is the way to go, if field of view, zoom, ok. - The Olympus software released in mid-April (2018) now is able to use NVidia GPU. Negotiate with your salespeople (i.e. Leica [SVI Huygens for HyVolution2, don't know if they also do their own thing for FALCON), Olympus, Nikon may also be GPU, Microvolution, AutoQuant) on price of the deconvolution AND what GPU card(s) is(are) going into the workstation (might end up being sold a cheap GPU and then going out and buying the right one ... make sure the case and power supply can handle it/them). Also important to know if the workstation can handle say 3 Quadro GV100's (three double width PCIe cards) or say 4 V100's on the motherboard, see for example, https://www.supermicro.com/products/system/4u/7048/sys-7048gr-tr.cfm (sure, one FV100 or one V100 is $9000, but one or more would enable 'instant gratification' on a possibly $500K confocal or $900K light sheet rig ... I also note that 100Gbe Ethernet cards are $800 so if your new PC quote is still 1gig, tell the salesperson to fix it ... sure, also need more cards, and probably a switch and 100Gbe NAS to send the data to). - Olympus "SSU" ultrasonic stage is very nice. If you go with Olympus, get it (or at least demo it and decide for yourself). You can ask other companies if they have the same thing. * I manage a Leica SP8 confocal microscope with 2 HyD detectors and HyVolution2, works great (I see I have more edits), http://confocal.jhu.edu/current-equipment/leica-sp8-confocal-microscope * looks like Nikon's spectral confocal unmixes in by fluorophore (finally a big confocal company gets it right), whereas Leica (LAS X on SP8, same as earlier LAS on SP5) and ZEN Black (which might or not be improved in 'zen one') unmix brightest channel first (bad idea if ever try to do controls, especially "fluorescence minus one" controls). * Geoff Daniels (Leica confocal sales) explained FALCON ... very impressive sounding. If you have the money, four FALCONhyD's should be great. If you have only money for one or two of them, start with that (the pulsed white light laser likely costs more than four HyDs). Ideally get an X1 port to enable more detectors (i.e. 2 or more APDs for NIR). * Zeiss LSM880 with Airyscan (I'm not sure why anyone would buy an 880 without Airyscan ... seems like a 780 and marketing added 100). * There are a lot of very interesting "other scopes" out there, mention three: - Abberior Instruments - see http://www.abberior-instruments.com/site/assets/files/1057/abberior_instruments_adaptive_optics_easy3d_sted_deep_imaging.png - ISS Alba and Alba-STED. - confocal.nl "rescan". - whatever AO LLS thingy Eric Betzig recently published ... hopefully whatever company eventually distributes it does not botch the product rollout. Note quite comprehensive table of microscope vendors is one of the tables in my (open access) Unit https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cphg.42 which is slightly newer than my online table http://www.geomcnamara.com/light-microscopy-websites enjoy, George p.s. if making "channel unmixing" matrix, I think photon counting is the way to go (HyD's), and do a lot of line and frame accumulation to get the best data for computing the coefficients (from single stained controls). For example, 16 line * 16 frame accumulations = 256 (the current max in leica LAS X), which produces bigger numbers than my experimental scanning 'standard of care' of 10 line accumulation (no frame accum). Sure, I am assuming no photobleaching (If bleaching was a concern, I would repeat, compare, and if no bleaching, add the two). On 5/10/2018 11:19 AM, Vitaly Boyko 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 Mike, > there is no big difference between HyDs and GaAsP detectors. Leica SMD HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more for the GaAsP.I am not aware of linear deconvolution algorithms for confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and not suitable for accurate intensity based image analysis.Recent integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new era to biology letting biologists quantify their data and image biological molecules at their physiological concentrations. However, most of biologists seems to be "scared" of basic equations...If you are located in the US, Leica provides much better Service and Application support compared to Zeiss. I do not have much experience with Olympus and Nikon on the confocal side. > If you have any specific questions, please let me know. > VitalyMCCF/MSKCC > On Thursday, May 10, 2018, 10:01:36 AM EDT, MODEL, MICHAEL <[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. > ***** > > Dear colleagues, > > > > we are looking for a new confocal microscope, and I have two questions so far. > > 1. Are Leica'a hybrid detectors (I am talking about SP8) in any way better than the standard GaAsP? I found an old discussion of this topic on this list, but technology may have improved since then. > > 2. Both Leica and Olympus 3000 use deconvolution to boost resolution. We haven't had enough time during the demos to test it thoroughly, but my impression is that some features "revealed" by deconvolution might be artefactual, so I don't know if it is worth paying extra. > > > > Thank you > > > > Mike Model > > Kent State University -- George McNamara, PhD Baltimore, MD 21231 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome) http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 http://confocal.jhu.edu July 2017 Current Protocols article, open access: UNIT 4.4 Microscopy and Image Analysis http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract supporting materials direct link is http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 figures at http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures |
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Vincent Schoonderwoert |
Addition to post on "confocal detectors and deconvolution"
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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. ***** ***Vendor message*** Dear all, Without getting involved in a discussion about the need for deconvolution (although we are tempted), we feel a profound need to inform you about the status of the collaboration between SVI-Huygens and Leica to prevent any misunderstanding if you are considering a new confocal microscope. Per December 1, 2017, despite its success, Leica stopped selling Hyvolution2 with Huygens as its computing engine. As George kindly mentioned, Hyvolution2 includes "the SVI Huygens software under the hood". New Leica customers are now offered another option in LASX that does NO LONGER involve the Huygens software and SVI. We will maintain our high level of support to customers who already purchased a Huygens license via Hyvolution or directly with their SP8. Like before, we also keep offering Huygens for the latest microscope types and file formats. Also the new and fully automated on-the-fly deconvolution tool "Batch Express" will offer this functionality. We welcome any further questions and will respond offline. Apologies to Mike for using your interesting post. With kind regards, Vincent Get the best out of your microscopy images with Huygens Batch Express: http://www.svi.nl/BatchExpress *********************************************** Vincent Schoonderwoert, PhD Senior Imaging Specialist/Account Manager Scientific Volume Imaging www.svi.nl +31 35 642 1626 *********************************************** |
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Cole, Richard W (HEALTH) |
confocal detectors and deconvolution
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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. ***** Mike, I have two HyDs and I agree w/Vitaly, there is essentially no difference when compared to GaAsP. We run almost all imaging modalities through deconvolution (Autoquant GPU), and yes there has to be some care taken so that artifacts (present in raw data, ie SA) are not accentuated. As for quantitative decon see: Calibration of wide-field deconvolution microscopy for quantitative fluorescence imaging. Lee JS; Wee TL; Brown CM. J. of Biomol. Tech. 25(1):31-40, 2014 Apr. My 2¢ Richard Cole Research Scientist V Director: Advanced Light Microscopy & Image Analysis Core Wadsworth Center Research Assistant Professor Dept. of Biomedical Sciences School of Public Health State University of New York 120 New Scotland Avenue, Albany N.Y. 12208 518-474-7048 Phone 518-408-1730 Fax >Hi Mike, >there is no big difference between HyDs and GaAsP detectors. Leica SMD HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more for the GaAsP.I am >not aware of linear deconvolution algorithms for confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and not suitable for accurate intensity based >image analysis.Recent integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new era to biology letting biologists quantify their data and image >biological molecules at their physiological concentrations. However, most of biologists seems to be "scared" of basic equations...If you are located in the US, Leica provides >much better Service and Application support compared to Zeiss. I do not have much experience with Olympus and Nikon on the confocal side. >If you have any specific questions, please let me know. >VitalyMCCF/MSKCC http://www.lsoft.com/products/listserv-powered.asp |
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Steffen Dietzel |
Re: confocal detectors and deconvolution
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In reply to this post by Vitaly Boyko-2
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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. ***** Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: > there is no big difference between HyDs and GaAsP detectors. I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. Steffen -- ------------------------------------------------------------ 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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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. ***** We have potential users who want to quantify some kind of small aggregates in the brain. I am afraid that deconvolution can make noise look like such aggregates. Perhaps collecting a noisy image twice and comparing two deconvolved images might help, but that seems too much work. Am I wrong? -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: Friday, May 11, 2018 10:49 AM To: [hidden email] Subject: Re: confocal detectors and deconvolution ***** 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. ***** Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: > there is no big difference between HyDs and GaAsP detectors. I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. Steffen -- ------------------------------------------------------------ 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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Zdenek Svindrych-2 |
Re: confocal detectors and deconvolution
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In reply to this post by Steffen Dietzel
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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 Steffen, the electron multiplication process is stochastic both in HyDs and regular PMTs. The difference is that HyDs are always used in photon counting mode (afaik), whereas PMTs (GaAsP or other) may or may not, depending on the instrument. HyDs should be able to handle higher photon rates than photon counting PMTs (at least that's what Leica used to say), while they have much bigger active area than SPADs, so they are easier to align. That being said, I'd like to see more SPADs on regular confocals... Best, zdenek ---------- Původní e-mail ---------- Od: Steffen Dietzel <[hidden email]> Komu: [hidden email] Datum: 11. 5. 2018 11:01:03 Předmět: Re: confocal detectors and deconvolution "***** 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. ***** Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: > there is no big difference between HyDs and GaAsP detectors. I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. Steffen -- ------------------------------------------------------------ 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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Alison J. North |
Re: Addition to post on "confocal detectors and deconvolution"
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In reply to this post by Vincent Schoonderwoert
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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 all, I am a big fan of carefully performed deconvolution, and we have used Huygens, Autoquant and GE's SoftWoRx deconvolution packages on many types of microscope images (widefield, confocal, spinning disk, light sheet etc.) with great success. However, I'm not sure I agree fully with George's comment that every confocal microscope should have deconvolution running immediately downstream, unless the manufacturers are going to do a better job of educating every single user about what goes on in their black box of deconvolution. As we all know, some deconvolution softwares are more quantitative than others. I am particularly concerned after a talk I just heard about the new Leica LIGHTNING software, which uses different parameters for each voxel in the image. Here is a direct quote from Leica's web page: "Maximize the information you extract from your specimen and get in-depth answers to scientific questions with the LIGHTNING detection package for image information extraction. LIGHTNING fully automatically detects the finest structures and details, which are otherwise simply not visible. The key technology of LIGHTNING is an adaptive process for extraction of hidden information in the image. Unlike traditional technologies, that use a global set of parameters for the full image, *LIGHTNING calculates an appropriate set of parameters for each voxel *to uncover every detail with the highest fidelity." I think we need to impress upon the microscope manufacturers very strongly that we are in the business of collecting quantitative scientific data, not just pretty pictures. At their presentation of this new software at the ABRF meeting last week, I asked whether it would be possible for them to flash up a big red warning on the screen - "Pretty picture ONLY!" - every time somebody uses this operation, to warn the user that this is a non-quantitative operation and therefore the user will never be able to perform any kind of quantitative image analysis on those images. I'm not sure they took my comment seriously, but I was indeed being perfectly serious! I also think the same should be implemented for any kind of operation that turns a quantitative raw image into one that is not, such as the use of the High Dynamic Range button on some of the confocal systems. Since many journals require authors to declare any nonlinear operations to images, including gamma adjustments, it's critical for every researcher to be aware of any such operations. It may be acceptable to perform this kind of operation after the fact, as long as one understands what has been done and states it clearly in the methods or figure legend, but I am very nervous about acquiring RAW data that has already been changed in a nonlinear way. I'm not meaning to pick on Leica - they are not the only ones with evil buttons and options in their software! - but I do believe we need to point out how worrying this trend is. Best, Alison On 5/11/2018 8:55 AM, Vincent Schoonderwoert 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=DwIDaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=otc_bgF6TO_iCl4lyz-BEkOTqW9EsRGuiTXpZHmRB3U&s=AR4nk9u2v55TucTfbtDFWmLZpC8zt70fMkhWmC9TtIQ&e= > > Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIDaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=otc_bgF6TO_iCl4lyz-BEkOTqW9EsRGuiTXpZHmRB3U&s=9p6_6YMvYBgHgIKwAUK46-MDCP_Rsm-V8WV0zaFIGE4&e= > and include the link in your posting. > ***** > > ***Vendor message*** > > > Dear all, > > > Without getting involved in a discussion about the need for > deconvolution (although we are tempted), we feel a profound need to > inform you about the status of the collaboration between SVI-Huygens > and Leica to prevent any misunderstanding if you are considering a new > confocal microscope. > > Per December 1, 2017, despite its success, Leica stopped selling > Hyvolution2 with Huygens as its computing engine. As George kindly > mentioned, Hyvolution2 includes "the SVI Huygens software under the > hood". New Leica customers are now offered another option in LASX that > does NO LONGER involve the Huygens software and SVI. > > We will maintain our high level of support to customers who already > purchased a Huygens license via Hyvolution or directly with their SP8. > Like before, we also keep offering Huygens for the latest microscope > types and file formats. Also the new and fully automated on-the-fly > deconvolution tool "Batch Express" will offer this functionality. > > We welcome any further questions and will respond offline. > > Apologies to Mike for using your interesting post. > > With kind regards, > Vincent > > > Get the best out of your microscopy images with Huygens Batch Express: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.svi.nl_BatchExpress&d=DwIDaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=otc_bgF6TO_iCl4lyz-BEkOTqW9EsRGuiTXpZHmRB3U&s=EAEoN_a12grsO5QrEbCKFIa1T8P_LjFnR_rzGasiUpk&e= > > > *********************************************** > Vincent Schoonderwoert, PhD > Senior Imaging Specialist/Account Manager > Scientific Volume Imaging > www.svi.nl > +31 35 642 1626 > *********************************************** -- 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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Brian Northan |
Re: confocal detectors and deconvolution
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In reply to this post by Cole, Richard W (HEALTH)
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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. ***** > > and yes there has to be some care taken so that artifacts (present in raw > data, ie SA) Hi Rich Artifacts in deconvolved images, caused by aberrated PSF, can be avoided to some extent, by using an accurate PSF. Either a high quality measured PSF, or (atleast for SA) a theoretical PSF (ie Gibson Lanni) with good estimates for Refractive Index of the Specimen, and Imaging depth. Mike wrote a paper, a few years ago, on PSF generation and deconvolving aberrated images (https://www.ncbi.nlm.nih.gov/pubmed/21118213). Brian On Fri, May 11, 2018 at 9:17 AM, Cole, Richard W (HEALTH) < [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. > ***** > > Mike, > > I have two HyDs and I agree w/Vitaly, there is essentially no difference > when compared to GaAsP. We run almost all imaging modalities through > deconvolution (Autoquant GPU), and yes there has to be some care taken so > that artifacts (present in raw data, ie SA) are not accentuated. As for > quantitative decon see: Calibration of wide-field deco > nvolution microscopy for quantitative fluorescence imaging. Lee JS; Wee > TL; Brown CM. J. of Biomol. Tech. 25(1):31-40, 2014 Apr. > > My 2¢ > > > Richard Cole > Research Scientist V > Director: Advanced Light Microscopy & Image Analysis Core > Wadsworth Center > > Research Assistant Professor > Dept. of Biomedical Sciences > School of Public Health State University of New York > > 120 New Scotland Avenue, Albany N.Y. 12208 > 518-474-7048 Phone > 518-408-1730 Fax > > > > > > >Hi Mike, > >there is no big difference between HyDs and GaAsP detectors. Leica SMD > HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more > for the GaAsP.I am >not aware of linear deconvolution algorithms for > confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and > not suitable for accurate intensity based >image analysis.Recent > integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new > era to biology letting biologists quantify their data and image >biological > molecules at their physiological concentrations. However, most of > biologists seems to be "scared" of basic equations...If you are located in > the US, Leica provides >much better Service and Application support > compared to Zeiss. I do not have much experience with Olympus and Nikon on > the confocal side. > >If you have any specific questions, please let me know. > >VitalyMCCF/MSKCC > > http://www.lsoft.com/products/listserv-powered.asp > On Fri, May 11, 2018 at 9:17 AM, Cole, Richard W (HEALTH) < [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. > ***** > > Mike, > > I have two HyDs and I agree w/Vitaly, there is essentially no difference > when compared to GaAsP. We run almost all imaging modalities through > deconvolution (Autoquant GPU), and yes there has to be some care taken so > that artifacts (present in raw data, ie SA) are not accentuated. As for > quantitative decon see: Calibration of wide-field > deconvolution microscopy for quantitative fluorescence imaging. Lee JS; > Wee TL; Brown CM. J. of Biomol. Tech. 25(1):31-40, 2014 Apr. > > My 2¢ > > > Richard Cole > Research Scientist V > Director: Advanced Light Microscopy & Image Analysis Core > Wadsworth Center > > Research Assistant Professor > Dept. of Biomedical Sciences > School of Public Health State University of New York > > 120 New Scotland Avenue, Albany N.Y. 12208 > 518-474-7048 Phone > 518-408-1730 Fax > > > > > > >Hi Mike, > >there is no big difference between HyDs and GaAsP detectors. Leica SMD > HyDs are cooled to 18 C, thus having ca. 400 dark counts versus 2K or more > for the GaAsP.I am >not aware of linear deconvolution algorithms for > confocal image data, thus it seems to be "cosmetic" and/or "aesthetic", and > not suitable for accurate intensity based >image analysis.Recent > integration of FALCON combined with FCS/RICS to the Leica SP8 may bring new > era to biology letting biologists quantify their data and image >biological > molecules at their physiological concentrations. However, most of > biologists seems to be "scared" of basic equations...If you are located in > the US, Leica provides >much better Service and Application support > compared to Zeiss. I do not have much experience with Olympus and Nikon on > the confocal side. > >If you have any specific questions, please let me know. > >VitalyMCCF/MSKCC > > http://www.lsoft.com/products/listserv-powered.asp > |
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Thomas Abraham |
Huygen Professional-SVI Question
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In reply to this post by Vincent Schoonderwoert
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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. ***** We have Huygens Pro (Ver 17.XX) in one our computer and that computer went down recently because of some hardware issues. We wanted to switch that version over to a new computer. When I contacted Huygens, they wanted so called “Migration Fee” of $399 for license string! Btw, we have two other commercial programs VOLOCITY and IMARIS. Both companies DO NOT charge anything for changing computers. In my view, this charge from HUYGENS is very unfair. The buyer, who already paid for the software, should decide where he/she wants to run the software. I would like to know any other companies have similar practices. Thanks in advance for your response. -------------------------------------------------------------- Thomas Abraham, PhD Director, Microscopy Imaging Core Lab Penn State College of Medicine Penn State Website: https://profiles.psu.edu/profiles/display/82782779 <https://profiles.psu.edu/profiles/display/82782779> ------------------------------------------------------------- > On May 11, 2018, at 8:55 AM, Vincent Schoonderwoert <[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. > ***** > > ***Vendor message*** > > > Dear all, > > > Without getting involved in a discussion about the need for deconvolution (although we are tempted), we feel a profound need to inform you about the status of the collaboration between SVI-Huygens and Leica to prevent any misunderstanding if you are considering a new confocal microscope. > > Per December 1, 2017, despite its success, Leica stopped selling Hyvolution2 with Huygens as its computing engine. As George kindly mentioned, Hyvolution2 includes "the SVI Huygens software under the hood". New Leica customers are now offered another option in LASX that does NO LONGER involve the Huygens software and SVI. > > We will maintain our high level of support to customers who already purchased a Huygens license via Hyvolution or directly with their SP8. Like before, we also keep offering Huygens for the latest microscope types and file formats. Also the new and fully automated on-the-fly deconvolution tool "Batch Express" will offer this functionality. > > We welcome any further questions and will respond offline. > > Apologies to Mike for using your interesting post. > > With kind regards, > Vincent > > > Get the best out of your microscopy images with Huygens Batch Express: > > http://www.svi.nl/BatchExpress > > *********************************************** > Vincent Schoonderwoert, PhD > Senior Imaging Specialist/Account Manager > Scientific Volume Imaging > www.svi.nl > +31 35 642 1626 > *********************************************** |
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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 agisoft photoscan for photogrammetry, and the licence is machine locked, but the software allows you to deactivate the licence on that machine, install on AN other and reactivate the icence on that new machine, all the functionality is built into the software itself, you can just move it around between PCs at will, without any need for human intervention at the supplier end. |
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Zdenek Svindrych-2 |
Re: Huygen Professional-SVI Question
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In reply to this post by Thomas Abraham
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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 Thomas, never happened to me, we used to run ancient version of the software on ancient hardware and we never had to migrate. The problem is that the software protection of Huygens is apparently very basic, and after they send you licence for the new machine, they have no way of stopping you from using the old one as well. They need to rely on your fairness and honesty... If you could only buy fairness for $399! Yes, I think it's unfair, and it does not look as good business practice. And it's us, (potential) customers who can change it. But beware, that one possible outcome is that SVI start using commercial software protection (e.g. USB dongles), that will make the software more expensive... Anyway, thanks for bringing this up. Btw, they do mention it as 'administrative fee' at their website ( svi.nl/ Maintenance ). Alternatively, you might try to negotiate a Maintenance contract, surely more expensive, but you could migrate AND get the newest version. Absolutely no commercial interest. Best, zdenek ---------- Původní e-mail ---------- Od: Thomas Abraham <[hidden email]> Komu: [hidden email] Datum: 12. 5. 2018 6:33:44 Předmět: Huygen Professional-SVI Question "***** 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. ***** We have Huygens Pro (Ver 17.XX) in one our computer and that computer went down recently because of some hardware issues. We wanted to switch that version over to a new computer. When I contacted Huygens, they wanted so called “Migration Fee” of $399 for license string! Btw, we have two other commercial programs VOLOCITY and IMARIS. Both companies DO NOT charge anything for changing computers. In my view, this charge from HUYGENS is very unfair. The buyer, who already paid for the software, should decide where he/she wants to run the software. I would like to know any other companies have similar practices. Thanks in advance for your response. -------------------------------------------------------------- Thomas Abraham, PhD Director, Microscopy Imaging Core Lab Penn State College of Medicine Penn State Website: https://profiles.psu.edu/profiles/display/82782779 <https://profiles.psu. edu/profiles/display/82782779> ------------------------------------------------------------- > On May 11, 2018, at 8:55 AM, Vincent Schoonderwoert <[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. > ***** > > ***Vendor message*** > > > Dear all, > > > Without getting involved in a discussion about the need for deconvolution status of the collaboration between SVI-Huygens and Leica to prevent any misunderstanding if you are considering a new confocal microscope. > > Per December 1, 2017, despite its success, Leica stopped selling Hyvolution2 with Huygens as its computing engine. As George kindly mentioned, Hyvolution2 includes "the SVI Huygens software under the hood". New Leica customers are now offered another option in LASX that does NO LONGER involve the Huygens software and SVI. > > We will maintain our high level of support to customers who already purchased a Huygens license via Hyvolution or directly with their SP8. Like before, we also keep offering Huygens for the latest microscope types and file formats. Also the new and fully automated on-the-fly deconvolution tool "Batch Express" will offer this functionality. > > We welcome any further questions and will respond offline. > > Apologies to Mike for using your interesting post. > > With kind regards, > Vincent > > > Get the best out of your microscopy images with Huygens Batch Express: > > http://www.svi.nl/BatchExpress > > *********************************************** > Vincent Schoonderwoert, PhD > Senior Imaging Specialist/Account Manager > Scientific Volume Imaging > www.svi.nl > +31 35 642 1626 > *********************************************** |
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James Pawley |
Re: confocal detectors and deconvolution
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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. ***** Hi all, I would like to Eecho Michael’s points. Because hybrid photodetectors have a very high gain (>>1,000x) in their first stage, they produce very little “excess noise” (also called Multiplicative Noise). As a result, it is possible to characterize their output as "25 photons detected” (although it might be safer to think of it as "25 photons detected this time" or ”25 +/- 5 photons."). Straight PMTs do not share this feature and because single photoelectrons produce output pulses that vary significantly in size, even the very best PMTs produce an uncertainty in the magnitude of the signal presented to the ADC that is at least 40% larger in relative terms than would be the case in the absence of this excess noise. On PMTs having electron multipliers optimized for other reasons (such as making them very small, like those in the 32-PMT linear arrays), the increase in uncertainty is closer to 100% (i.e., The signal has the same uncertainty that it would have if 4 times fewer photons were counted perfectly.) Either type of PMT can have a GaAsP photocathode but it will need to be cooled. Although single APDs may have a high photon detection efficiency (PDE, a spec that is like QE but which includes the signal lost by photoelectrons that do not avalanche at all) they have such massive excess noise that it is essential to use them with pulse-counting circuits and these circuits are just too slow for use in beam-scanning light microscopy. The solution is the multi-pixel photon counter (MPPC, a development from the SPAD (single photon avalanche device), https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_ultimate_photosensor/index.html ). The surface of an MPPC is covered with an array of 400 to 20,000 APDs, each connected to the high-voltage rail through its own damping resister. The resistor causes the voltage across the APD to drop as the avalanche proceeds. This quenches the discharge and produces single-photon pulses of very uniform size. As all the APDs are electrically in parallel, these single-photon current pulses simply add up producing an output current signal almost devoid of excess noise. What could be better? And in addition they are significantly less sensitive than hybrid PMTs to overheating damage if accidentally exposed to a bright light. There are of course limitations: 1) A significant fraction (20-40%) of the MPPC's surface is taken up with the resistors and the wiring to provide each APD with + and - voltages. Photons absorbed or reflected in these areas are lost. 2) The system is only free of pulse-pileup losses to the extent that no APD absorbs more than one photon within its RC relaxation time (set by the R of the resistor, and the capacitance (C) of the sensitive area of the APD. Larger individual APDs “waste” proportionally fewer photons hitting the resistor and wiring, (increasing their effective QE) but this increases their capacitance (making them more susceptible to pulse-pileup). All will be well as long as the number of photons absorbed in the active areas during time period RC is small (5%?) compared to the number of APDs in the array THAT ARE ILLUMINATED BY THE BEAM. The caps above are to remind everyone that, to work properly, the size of the ray bundle striking the MPPC must be matched to the size of the APD array (1.3 to 6 mm square). This can be a problem if we imagine the ray bundle being limited by a confocal aperture that can be varied in size over a substantial range. (Do we need a zoom lens to make all possible signal ray-bundles match the size of the MPPC array?) Apart from this, I would like to second the comment that deep imaging is usually limited by spherical aberration and systems that can correct for this without “bumping the specimen while you try to adjust the collar” are to be preferred. I would also like to reaffirm that, assuming the pixel size meets Nyquist, you should ONLY evaluate results after deconvolving the data with an appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist-sampled image will be at least 4 (more likely 5) pixels wide, all the noise terms affect single pixel values. i.e., they have frequency components at least 4x higher than that which can represent any real structure. In scanned fluorescent imaging, one deconvolves more to reduce noise than to increase “spatial resolution” (although you can also increase resolution as long as you have massive amounts of signal.) Cheers, Jim Pawley **************************************** James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[hidden email]<mailto:[hidden email]>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146 On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:[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. ***** We have potential users who want to quantify some kind of small aggregates in the brain. I am afraid that deconvolution can make noise look like such aggregates. Perhaps collecting a noisy image twice and comparing two deconvolved images might help, but that seems too much work. Am I wrong? -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: Friday, May 11, 2018 10:49 AM To: [hidden email] Subject: Re: confocal detectors and deconvolution ***** 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. ***** Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: there is no big difference between HyDs and GaAsP detectors. I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. Steffen -- ------------------------------------------------------------ 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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George McNamara |
Re: confocal detectors and deconvolution
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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. ***** Hi Mike, Collect two 'as clean as possible' images, deconvolve and compare those. I have occasionally acquired on our HyDs: line accumulation: 16 frame accumulation: 16 (both max'd out ... I would like Leica to enable the drop down lists to go to 100, or even 256, instead of max 16 ... I manage a core that charges by the hour, so more accum --> more revenue <--- better data). Pinhole 0.5 Airy Unit (the graphs I have in the SP8 room -- from some Zeiss pdf -- show some more improvement in XY if go to 0.28 airy unit, for sparse features this would be ~0.3^2 = 0.09 vs ~0.5^2 = 0.25, photons getting through the pupil than 1 A.U. 63x/1.4NA objective, 40 nm XY pixel size, 120 nm Z. Modest laser powers (yes, some photobleaching, usually). I acquire in 16-bit mode even if doing only a few line accumulation and frame accumulation = 1. 'Standard' fluorophores (i.e. Alexa Fluor 488) and mounting media (mostly Prolong Gold with the 1.4NA lens). Yes, would be better with better fluorophore (i.e. Janelia Fluors, maybe BD Brilliants, Thermo's SuperBrights, more) and optimal R.I. matching per specimen and objective lens. * Then try out a couple of deconvolution settings wrt SVI's options in HyVolution2. On GaAsP PMT, optimized gain (and offset), optimized choice(s) of summing and/or averaging (i.e. 16-bit mode). George p.s. Allison - great post about us wanting quantitative devices and the companies should stop pushing 'a pretty picture'. Memo to confocal companies: if we just wanted pretty pictures, we would be running cRAYon Distribution cores. On 5/11/2018 11:16 AM, MODEL, MICHAEL 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. > ***** > > We have potential users who want to quantify some kind of small aggregates in the brain. I am afraid that deconvolution can make noise look like such aggregates. Perhaps collecting a noisy image twice and comparing two deconvolved images might help, but that seems too much work. Am I wrong? > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel > Sent: Friday, May 11, 2018 10:49 AM > To: [hidden email] > Subject: Re: confocal detectors and deconvolution > > ***** > 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. > ***** > > Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: >> there is no big difference between HyDs and GaAsP detectors. > I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. > > Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. > > As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. > > Steffen > > -- > ------------------------------------------------------------ > 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 -- George McNamara, PhD Baltimore, MD 21231 [hidden email] https://www.linkedin.com/in/georgemcnamara https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome) http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650 http://confocal.jhu.edu July 2017 Current Protocols article, open access: UNIT 4.4 Microscopy and Image Analysis http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract supporting materials direct link is http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023 figures at http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures |
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Gitta Hamel |
Re: Huygen Professional-SVI Question
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In reply to this post by Thomas Abraham
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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. ***** **Vendor response** Dear Thomas, We fully understand that maintenance and upgrade fees can be a burden in your facility. We regret the confusion that the offer may have caused. The /migration only / offer was sent after a/maintenanc//e/ proposal was rejected for very good reasons that we fully respect. Accompanying the /migration only/ offer was the statement that this fee would be /100//% deducted/ if you would decide to accept the maintenance proposal. At SVI we offer a lotwith a maintenance and upgrade contract as it includes: Twice a year a major upgrade, as well as fast bug fixes resulting in updated version every couple of weeks to facilitate our users to benefit from the bug fixes as soon as possible. In your Huygens license you see the End of Maintenance date written out. Also included is a free of charge migration /once a year/ while under maintenance. Next to that we offer fast responses on support questions we receive, even during the weekend. You experienced our support, when you expressed a need to work with Huygens this Saturday and we supplied you with a fully functional Huygens license the same day. We very much hope that the experiments were successful? With best wishes, Gitta Hamel ****************************** Gitta Hamel Managing Director/Imaging Specialist Scientific Volume Imaging bv Laapersveld 63 1213 VB Hilversum The Netherlands www.svi.nl [hidden email] [hidden email] tel: ++ 31 35 6 42 16 26 Cell:++ 31 6 18 02 12 72 ****************************** On 12-05-18 12:29, Thomas Abraham 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. > ***** > > We have Huygens Pro (Ver 17.XX) in one our computer and that computer went down recently because of some hardware issues. We wanted to switch that version over to a new computer. When I contacted Huygens, they wanted so called “Migration Fee” of $399 for license string! Btw, we have two other commercial programs VOLOCITY and IMARIS. Both companies DO NOT charge anything for changing computers. In my view, this charge from HUYGENS is very unfair. The buyer, who already paid for the software, should decide where he/she wants to run the software. I would like to know any other companies have similar practices. Thanks in advance for your response. > > -------------------------------------------------------------- > Thomas Abraham, PhD > Director, Microscopy Imaging Core Lab > Penn State College of Medicine > Penn State Website: > https://profiles.psu.edu/profiles/display/82782779 <https://profiles.psu.edu/profiles/display/82782779> > ------------------------------------------------------------- > > > >> On May 11, 2018, at 8:55 AM, Vincent Schoonderwoert <[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. >> ***** >> >> ***Vendor message*** >> >> >> Dear all, >> >> >> Without getting involved in a discussion about the need for deconvolution (although we are tempted), we feel a profound need to inform you about the status of the collaboration between SVI-Huygens and Leica to prevent any misunderstanding if you are considering a new confocal microscope. >> >> Per December 1, 2017, despite its success, Leica stopped selling Hyvolution2 with Huygens as its computing engine. As George kindly mentioned, Hyvolution2 includes "the SVI Huygens software under the hood". New Leica customers are now offered another option in LASX that does NO LONGER involve the Huygens software and SVI. >> >> We will maintain our high level of support to customers who already purchased a Huygens license via Hyvolution or directly with their SP8. Like before, we also keep offering Huygens for the latest microscope types and file formats. Also the new and fully automated on-the-fly deconvolution tool "Batch Express" will offer this functionality. >> >> We welcome any further questions and will respond offline. >> >> Apologies to Mike for using your interesting post. >> >> With kind regards, >> Vincent >> >> >> Get the best out of your microscopy images with Huygens Batch Express: >> >> http://www.svi.nl/BatchExpress >> >> *********************************************** >> Vincent Schoonderwoert, PhD >> Senior Imaging Specialist/Account Manager >> Scientific Volume Imaging >> www.svi.nl >> +31 35 642 1626 >> *********************************************** |
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Brian Northan |
Re: Addition to post on "confocal detectors and deconvolution"
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In reply to this post by Alison J. North
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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 Allison You raise some really good points. I've been developing image analysis algorithms for 15+ years, and I'm always suspect of marketing, that has a lot of vague, "feel good", and subjective terms. The description of an image processing algorithm, should convey enough information, for example algorithms names and references, so that someone with a background in signal processing, can do a little research and understand what the algorithm does. As we all know, some deconvolution softwares are more quantitative than > others. As far as I am aware, little work has been done in this area. Especially in terms of evaluating morphology of results, and PSF models. What is needed is for a group of people, to evaluate the properties of the fundamental implementations of deconvolution and PSF generation algorithms (reference implementations are available from a variety of open sources such as DeconvolutionLab2, http://bigwww.epfl.ch/deconvolution/deconvolutionlab2/) and then evaluate whether Vendor implementations have the same properties. Going back to the "Lightning" product, using descriptive algorithm terminology and references, to describe the product, would go a long way to helping people understand and evaluate it properly. According to an answer on Leica's facebook page, it seems Lightning does use deconvolution,( https://www.facebook.com/LeicaMicrosystems/photos/a.145939838807420. 34521.135295379871866/1848698365198217/?type=3) As an algorithm developer I would be curious, exactly what they are doing. Are they doing multiple overlapping deconvolutions, with different parameters and blending them?? Are they using an spatially varying PSF?? Are they perhaps using recent techniques in machine learning (deep learning is a hot topic these days) to further enhance the image?? I don't really know, and can't begin to even try to understand what they are doing, based on the vague marketing terminology used on their web site. I think promoting a culture, where more importance is placed on the technical names, details and properties of algorithms would help in addressing some of these issues. If users of image processing software, can be educated on more of the nuts of bolts of image processing, it would help them ask vendors the right questions, and put pressure on vendors to give descriptive documentation Brian On Fri, May 11, 2018 at 9:35 AM, Alison North <[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. > ***** > > Dear all, > > I am a big fan of carefully performed deconvolution, and we have used > Huygens, Autoquant and GE's SoftWoRx deconvolution packages on many types > of microscope images (widefield, confocal, spinning disk, light sheet etc.) > with great success. However, I'm not sure I agree fully with George's > comment that every confocal microscope should have deconvolution running > immediately downstream, unless the manufacturers are going to do a better > job of educating every single user about what goes on in their black box of > deconvolution. As we all know, some deconvolution softwares are more > quantitative than others. I am particularly concerned after a talk I just > heard about the new Leica LIGHTNING software, which uses different > parameters for each voxel in the image. Here is a direct quote from > Leica's web page: > > "Maximize the information you extract from your specimen and get in-depth > answers to scientific questions with the LIGHTNING detection package for > image information extraction. > > LIGHTNING fully automatically detects the finest structures and details, > which are otherwise simply not visible. The key technology of LIGHTNING is > an adaptive process for extraction of hidden information in the image. > Unlike traditional technologies, that use a global set of parameters for > the full image, *LIGHTNING calculates an appropriate set of parameters for > each voxel *to uncover every detail with the highest fidelity." > > I think we need to impress upon the microscope manufacturers very strongly > that we are in the business of collecting quantitative scientific data, not > just pretty pictures. At their presentation of this new software at the > ABRF meeting last week, I asked whether it would be possible for them to > flash up a big red warning on the screen - "Pretty picture ONLY!" - every > time somebody uses this operation, to warn the user that this is a > non-quantitative operation and therefore the user will never be able to > perform any kind of quantitative image analysis on those images. I'm not > sure they took my comment seriously, but I was indeed being perfectly > serious! I also think the same should be implemented for any kind of > operation that turns a quantitative raw image into one that is not, such as > the use of the High Dynamic Range button on some of the confocal systems. > Since many journals require authors to declare any nonlinear operations to > images, including gamma adjustments, it's critical for every researcher to > be aware of any such operations. It may be acceptable to perform this kind > of operation after the fact, as long as one understands what has been done > and states it clearly in the methods or figure legend, but I am very > nervous about acquiring RAW data that has already been changed in a > nonlinear way. > > I'm not meaning to pick on Leica - they are not the only ones with evil > buttons and options in their software! - but I do believe we need to point > out how worrying this trend is. > > Best, > Alison > > > > On 5/11/2018 8:55 AM, Vincent Schoonderwoert wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.um >> n.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIDaQ&c=JeTk >> UgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNm >> FbqYvikvIZS5ns3-USwvMOuLo&m=otc_bgF6TO_iCl4lyz-BEkOTqW9EsR >> GuiTXpZHmRB3U&s=AR4nk9u2v55TucTfbtDFWmLZpC8zt70fMkhWmC9TtIQ&e= >> Post images on https://urldefense.proofpoint. >> com/v2/url?u=http-3A__www.imgur.com&d=DwIDaQ&c=JeTkUgVztGMmh >> KYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvI >> ZS5ns3-USwvMOuLo&m=otc_bgF6TO_iCl4lyz-BEkOTqW9EsRGuiTXpZHmRB >> 3U&s=9p6_6YMvYBgHgIKwAUK46-MDCP_Rsm-V8WV0zaFIGE4&e= and include the link >> in your posting. >> ***** >> >> ***Vendor message*** >> >> >> Dear all, >> >> >> Without getting involved in a discussion about the need for deconvolution >> (although we are tempted), we feel a profound need to inform you about the >> status of the collaboration between SVI-Huygens and Leica to prevent any >> misunderstanding if you are considering a new confocal microscope. >> >> Per December 1, 2017, despite its success, Leica stopped selling >> Hyvolution2 with Huygens as its computing engine. As George kindly >> mentioned, Hyvolution2 includes "the SVI Huygens software under the hood". >> New Leica customers are now offered another option in LASX that does NO >> LONGER involve the Huygens software and SVI. >> >> We will maintain our high level of support to customers who already >> purchased a Huygens license via Hyvolution or directly with their SP8. Like >> before, we also keep offering Huygens for the latest microscope types and >> file formats. Also the new and fully automated on-the-fly deconvolution >> tool "Batch Express" will offer this functionality. >> >> We welcome any further questions and will respond offline. >> >> Apologies to Mike for using your interesting post. >> >> With kind regards, >> Vincent >> >> >> Get the best out of your microscopy images with Huygens Batch Express: >> >> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.svi. >> nl_BatchExpress&d=DwIDaQ&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxX >> ez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m= >> otc_bgF6TO_iCl4lyz-BEkOTqW9EsRGuiTXpZHmRB3U&s=EAEoN_ >> a12grsO5QrEbCKFIa1T8P_LjFnR_rzGasiUpk&e= >> >> *********************************************** >> Vincent Schoonderwoert, PhD >> Senior Imaging Specialist/Account Manager >> Scientific Volume Imaging >> www.svi.nl >> +31 35 642 1626 >> *********************************************** >> > > -- > Alison J. North, Ph.D., > Research Associate Professor and > Senior Director of the Bio-Imaging Resource Ce > <https://maps.google.com/?q=irector+of+the+Bio-Imaging+Resource+Ce&entry=gmail&source=g> > nter, > 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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Zdenek Svindrych-2 |
Re: confocal detectors and deconvolution
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In reply to this post by James Pawley
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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 chill down the excitement around MCCPs (or SiPMs, as Hamamtsu call them), note that their dark noise is some 3 - 4 orders of magnitude higher than that of regular PMTs! Of course, chilling the detector to -80 degC (which is common with EMCCDs, for example) would solve this issue, but the cost would be prohibitive... For more details on SiPMs vs PMTs see here: https://drive.google.com/open?id=1R3hDR0KX0nE5qWh5GrzyELzBoiGdP1MB Cheers, zdenek ---------- Původní e-mail ---------- Od: JAMES B PAWLEY <[hidden email]> Komu: [hidden email] Datum: 14. 5. 2018 0:27:35 Předmět: Re: confocal detectors and deconvolution "***** 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, I would like to Eecho Michael’s points. Because hybrid photodetectors have a very high gain (>>1,000x) in their first stage, they produce very little “excess noise” (also called Multiplicative Noise). As a result, it is possible to characterize their output as "25 photons detected” (although it might be safer to think of it as "25 photons detected this time" or ”25 +/- 5 photons."). Straight PMTs do not share this feature and because single photoelectrons produce output pulses that vary significantly in size, even the very best PMTs produce an uncertainty in the magnitude of the signal presented to the ADC that is at least 40% larger in relative terms than would be the case in the absence of this excess noise. On PMTs having electron multipliers optimized for other reasons (such as making them very small, like those in the 32-PMT linear arrays), the increase in uncertainty is closer to 100% (i. e., The signal has the same uncertainty that it would have if 4 times fewer photons were counted perfectly.) Either type of PMT can have a GaAsP photocathode but it will need to be cooled. Although single APDs may have a high photon detection efficiency (PDE, a spec that is like QE but which includes the signal lost by photoelectrons that do not avalanche at all) they have such massive excess noise that it is essential to use them with pulse-counting circuits and these circuits are just too slow for use in beam-scanning light microscopy. The solution is the multi-pixel photon counter (MPPC, a development from the SPAD (single photon avalanche device), https://www.hamamatsu.com/resources/pdf/ssd/mppc_kapd0004e.pdf https://www.hamamatsu.com/us/en/community/optical_sensors/articles/sipm_the_ ultimate_photosensor/index.html ). The surface of an MPPC is covered with an array of 400 to 20,000 APDs, each connected to the high-voltage rail through its own damping resister. The resistor causes the voltage across the APD to drop as the avalanche proceeds. This quenches the discharge and produces single-photon pulses of very uniform size. As all the APDs are electrically in parallel, these single-photon current pulses simply add up producing an output current signal almost devoid of excess noise. What could be better? And in addition they are significantly less sensitive than hybrid PMTs to overheating damage if accidentally exposed to a bright light. There are of course limitations: 1) A significant fraction (20-40%) of the MPPC's surface is taken up with the resistors and the wiring to provide each APD with + and - voltages. Photons absorbed or reflected in these areas are lost. 2) The system is only free of pulse-pileup losses to the extent that no APD absorbs more than one photon within its RC relaxation time (set by the R of the resistor, and the capacitance (C) of the sensitive area of the APD. Larger individual APDs “waste” proportionally fewer photons hitting the resistor and wiring, (increasing their effective QE) but this increases their capacitance (making them more susceptible to pulse-pileup). All will be well as long as the number of photons absorbed in the active areas during time period RC is small (5%?) compared to the number of APDs in the array THAT ARE ILLUMINATED BY THE BEAM. The caps above are to remind everyone that, to work properly, the size of the ray bundle striking the MPPC must be matched to the size of the APD array (1.3 to 6 mm square). This can be a problem if we imagine the ray bundle being limited by a confocal aperture that can be varied in size over a substantial range. (Do we need a zoom lens to make all possible signal ray -bundles match the size of the MPPC array?) Apart from this, I would like to second the comment that deep imaging is usually limited by spherical aberration and systems that can correct for this without “bumping the specimen while you try to adjust the collar” are to be preferred. I would also like to reaffirm that, assuming the pixel size meets Nyquist, you should ONLY evaluate results after deconvolving the data with an appropriate 2D or 3D PSF. Although the smallest real object in a Nyquist- sampled image will be at least 4 (more likely 5) pixels wide, all the noise terms affect single pixel values. i.e., they have frequency components at least 4x higher than that which can represent any real structure. In scanned fluorescent imaging, one deconvolves more to reduce noise than to increase “ spatial resolution” (although you can also increase resolution as long as you have massive amounts of signal.) Cheers, Jim Pawley **************************************** James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[hidden email]<mailto:[hidden email]>> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146 On May 11, 18, at 10:16 AM, MODEL, MICHAEL <[hidden email]<mailto:mmodel@ KENT.EDU>> 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. ***** We have potential users who want to quantify some kind of small aggregates in the brain. I am afraid that deconvolution can make noise look like such aggregates. Perhaps collecting a noisy image twice and comparing two deconvolved images might help, but that seems too much work. Am I wrong? -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: Friday, May 11, 2018 10:49 AM To: [hidden email] Subject: Re: confocal detectors and deconvolution ***** 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. ***** Am 10.05.2018 um 17:19 schrieb Vitaly Boyko: there is no big difference between HyDs and GaAsP detectors. I disagree on this. In my view, the major difference is that the HyD always operates in photon counting mode whether, as far as I know, the PMTs (with or without GaAsP) create an electron cloud of which the size is determined by the number of photoelectrons AND statistics, and the cloud size is then digitized. So the output created by one photon may vary substantially depending on the number of electrons created on the first dynodes (which in turn is a statistical process). My information may be outdated and newer PMTs might have extra tricks, if so please correct me. Another difference is apparently the size of the photcathode. If memory serves me right, the larger cathode of the GaAsP PMTs (compared to HyDs) creates more dark noise. I like our HyDs a lot, I appreciate having a gray value of "21 photons" instead of some random number. But having said this, at the end of the day what counts is the sensitivity of the whole system, and not of the detector alone. So to do this right there is no substitute for testing your own samples on different machines with your applications in mind. As for deconvolution, yes, it can create artefacts. But so does confocal microscopy (a point becomes an Airy pattern, not a point). And if you do it right the deconvolved image will be closer to the truth than the original image. Should you have the third edition of the handbook around, have a look at the preface, last paragraph. Steffen -- ------------------------------------------------------------ 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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Steffen Dietzel |
Re: confocal detectors and deconvolution
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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. ***** Am 14.05.2018 um 07:19 schrieb George McNamara: > line accumulation: 16 > > frame accumulation: 16 > > (both max'd out ... I would like Leica to enable the drop down lists > to go to 100, or even 256, instead of max 16 ... I manage a core that > charges by the hour, so more accum --> more revenue <--- better data). George, the frame accumulation I suggest to substitute with a time series and making the sum of the images. This also gives a nice area to play around, like: how many images you need until you can't see improvement any more, or how random are results when images are compared. Or what your stage drifting speed is... Concerning the revenue, maybe you can convince the users that for optimal images a break of X minutes between the recordings is needed, so that the laser photons can refuel themselves :-) Steffen -- ------------------------------------------------------------ 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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