sCMOS salt'n'pepper issues
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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 hope the science/optics/hardware/software/students/post-docs/reviewers/managers/next-door-neighbors and FedEx are treating you well. We have a couple of issues with a recently installed Hamamatsu Flash 4.0 sCMOS camera and I was wondering if anybody has had any similar experiences, both in terms of feedback on what is expected from sCMOS hardware and what can be done to rectify any issues. Here's what we're seeing: Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're having three issues regarding hot and cold pixels. So far we have tried 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin Elmer), to double check the subtleties, but the results are quite similar On the first camera we saw a bright cluster of 4 pixels that are between 2 and 10x greater than the surrounding pixels across nearly all imaging conditions (not seen in the other three cameras). Are these bright pixel clusters something that others have seen much? This issue is seen in both Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used to cross check. I also see 'ghost' clusters that look similar but only seem to last for one frame... Again, is that something seen in sCMOS cameras in general? Salt: In images with low signal and longer exposure times we see speckled bight pixels using both Volocity and ExCap/HDImage, with an intensity approx twice that of the background signal. Exposure times ~secs; low signal <1000 counts per pixel. These speckled hot spots were seen with all sCMOS cameras to some degree. Pepper: In almost the inverse situation, where we have higher signals with lower exposure times were also seen on all sCMOS cameras (the first camera with the clear hotspot had very bad 'peppering' compared to the others). I know that the pco cameras used on the Deltavision OMX have reference images applied to reduce the affects of these artifacts, and I was wondering if that's something that can be applied post-acquisition. Any thoughts and/or feedback on what's normal for an sCMOS, and how to get the best out of these cameras would be much appreciated. sCMOS cameras certainly are fast and have a great signal to noise, so am I expecting too much from this generation of hardware? Thanks Neil A couple of images to compare salt'n'pepper pixels seen on three cameras are here: https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/view?usp=sharing https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/view?usp=sharing |
 
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Kyle Michael Douglass |
Re: sCMOS salt'n'pepper issues
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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 Neil, On 01/21/2015 04:24 AM, Neil Anthony wrote: > > On the first camera we saw a bright cluster of 4 pixels that are > between 2 and 10x greater than the surrounding pixels across nearly > all imaging conditions (not seen in the other three cameras). > We have an Andor Zyla 4.2 and I've tested a separate Zyla and the Flash 4.0 and I've never observed the clustering of hot spots. > Salt: > In images with low signal and longer exposure times we see speckled > bight pixels using both Volocity and ExCap/HDImage, with an intensity > approx twice that of the background signal. These hot spots I believe come from the "anonymously" noisy chip-level amplifiers. In an sCMOS camera, each pixel has its own amplifier and it seems that it's nearly impossible to ensure that all ~4 million pixels are defect free. In contrast, an EMCCD does not have pixel-dependent noise properties because the amplification is not pixel-dependent. Therefore, it seems like you have to accept hot spots by thinking of them as manufacturing defects that occur in making a large number of single pixels. We characterized our camera by measuring the pixel-dependent gain, noise variance, and hot spot locations according to the procedure in the supplement of this paper from the Bewersdorf lab: http://www.ncbi.nlm.nih.gov/pubmed/23708387. We found that our characterization matched the hot pixel map provided by Andor exactly, so we know what the intensity statistics of those pixels are. > Pepper: > In almost the inverse situation, where we have higher signals with > lower exposure times were also seen on all sCMOS cameras (the first > camera with the clear hotspot had very bad 'peppering' compared to the > others). I'm not sure about this one. I haven't noticed the peppering. Is the hot spot correction algorithm on in this case? > > I know that the pco cameras used on the Deltavision OMX have reference > images applied to reduce the affects of these artifacts, and I was > wondering if that's something that can be applied post-acquisition. We work in STORM/PALM microscopy and incorporate the hot spots into our noise model when performing the localization analysis, much like in the paper I cited above. We therefore do not use the manufactuer's algorithm for hot pixel smoothing. For "normal" imaging, I think you either have to use the hot spot correction algorithms to smooth over those pixels, or turn off the algorithm and post-process the hot spots yourself. I hope this helps! Kyle -- Kyle M. Douglass, PhD Post-doctoral researcher The Laboratory of Experimental Biophysics EPFL, Lausanne, Switzerland http://kmdouglass.github.io http://leb.epfl.ch |
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In reply to this post by Neil Anthony
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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. ***** Can you share raw data? Also, how are you doing your illumination? On Jan 20, 2015 11:19 PM, "Neil Anthony" <[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 all, I hope the science/optics/hardware/software/students/post-docs/ > reviewers/managers/next-door-neighbors and FedEx are treating you well. > > We have a couple of issues with a recently installed Hamamatsu Flash 4.0 > sCMOS camera and I was wondering if anybody has had any similar > experiences, both in terms of feedback on what is expected from sCMOS > hardware and what can be done to rectify any issues. > > Here's what we're seeing: > Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a > spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're > having three issues regarding hot and cold pixels. So far we have tried 4 > different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin > Elmer), to double check the subtleties, but the results are quite similar > > On the first camera we saw a bright cluster of 4 pixels that are between 2 > and 10x greater than the surrounding pixels across nearly all imaging > conditions (not seen in the other three cameras). Are these bright pixel > clusters something that others have seen much? This issue is seen in both > Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used > to cross check. I also see 'ghost' clusters that look similar but only > seem to last for one frame... Again, is that something seen in sCMOS > cameras in general? > > Salt: > In images with low signal and longer exposure times we see speckled bight > pixels using both Volocity and ExCap/HDImage, with an intensity approx > twice that of the background signal. Exposure times ~secs; low signal <1000 > counts per pixel. These speckled hot spots were seen with all sCMOS > cameras to some degree. > Pepper: > In almost the inverse situation, where we have higher signals with lower > exposure times were also seen on all sCMOS cameras (the first camera with > the clear hotspot had very bad 'peppering' compared to the others). > > I know that the pco cameras used on the Deltavision OMX have reference > images applied to reduce the affects of these artifacts, and I was > wondering if that's something that can be applied post-acquisition. Any > thoughts and/or feedback on what's normal for an sCMOS, and how to get the > best out of these cameras would be much appreciated. sCMOS cameras > certainly are fast and have a great signal to noise, so am I expecting too > much from this generation of hardware? > > Thanks > Neil > > > A couple of images to compare salt'n'pepper pixels seen on three cameras > are here: > https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/ > view?usp=sharing > https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/ > view?usp=sharing > |
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Christian Soeller |
Re: sCMOS salt'n'pepper issues
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In reply to this post by Neil Anthony
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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 Neil, We also have a Zyla 4.2 and I can mostly confirm Kyle's observation. > > Salt: > In images with low signal and longer exposure times we see speckled > bight pixels using both Volocity and ExCap/HDImage, with an intensity > approx twice that of the background signal. Exposure times ~secs; low > signal <1000 counts per pixel. These speckled hot spots were seen with > all sCMOS cameras to some degree. > > > The salt (white pixels I presume) you see may be related to pixels with higher dark currents which may become troubling for long exposures. This makes those pixels not great for long exposures. > Pepper: > In almost the inverse situation, where we have higher signals with lower > exposure times were also seen on all sCMOS cameras (the first camera > with the clear hotspot had very bad 'peppering' compared to the others). > The pepper, presumably very dark appearing pixels, could be due to at least two things. (1) a local low gain (gain is in principle pixel dependent) and (2) high read noise. A data post processing step is likely the best way to deal with some of these issues as are careful camera characterisation to guide that processing (see earlier reference to the Bewersdorf paper). Has anybody done this for confocal yet (rather than PALM/STORM)? In diffraction-limited imaging with suitable oversampling interpolation of missing data should be pretty doable. Do the manufacturers offer any assistance with this? I would have thought they should if they market these cameras as an option in these systems. Best, Christian |
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In reply to this post by Neil Anthony
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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 Neil, You did proper measurements and your findings are valid for sCMOS image sensors. It is very similar to the improvements in CCD image sensors before. Whenever the general noise behavior had been significantly improved, just in the lower noise signal additional noise phenomena became visible, that were not visible before, because they were hidden in the overall larger noise signal. It is definitively a image sensor issue, which can be seen in cameras of all the manufacturers. The scientific CMOS have become very sensitive, very low noise, but still they are CMOS image sensors, with dark current, with traps in the silicon, with electrical connections at the surface. Yes, both the "higher" noise pixel in the dark as well as the "darker" pixel in the bright are part of the "blinker" and higher noise pixels of sCMOS. Sometimes, if the noise histograms are given with a logarithmic scale, you will see that there is a significant tail towards the larger noise pixels, which might be improved in the future by Fairchild by changes in the production process, which is a complex task due to the many knobs that could be turned. In the dark, the amount of these guys will increase a little if the exposure time becomes very long, above 1 s. There you will see an increased offset value in some pixels, if you do an average measurement of a large stack of dark images, you will be able to see and detect the position of these pixels, which have not been put into the "hot pixel list" of the manufacturer. You could store their locations, and treat them either as defect pixels, try to use some kind of a weighted "salt-and-pepper" filtering or you do your own offset calibration in post processing. But these positions will not change, like the clock induced charge signals in emCCD for example. Due to the high gain, keep in mind, that there only few electrons involved. In the bright, there are some gain issues with some of the higher noise pixels. Again you can detect them, store the location and treat them as defect pixel or create your own additional gain calibration. Also here I would use a larger stack of homogenously illuminated medium bright images. You would need the averaging to exclude the photon noise. In general, the sCMOS has nice performance parameters, but in my opinion it is not optimum for long exposures. Its is better for higher frame rates and shorter exposure times, where the dark current and its noise influence can be neglected. with best regards, Gerhard _______________________________ Dr. Gerhard Holst Science & Research PCO AG Donaupark 11 93309 Kelheim, Germany fon +49 (0)9441 2005 36 fax +49 (0)9441 2005 20 mob +49 (0)172 711 6049 [hidden email] www.pco.de -----Ursprüngliche Nachricht----- Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Neil Anthony Gesendet: Mittwoch, 21. Januar 2015 04:24 An: [hidden email] Betreff: sCMOS salt'n'pepper issues ***** 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 hope the science/optics/hardware/software/students/post-docs/reviewers/managers/next-door-neighbors and FedEx are treating you well. We have a couple of issues with a recently installed Hamamatsu Flash 4.0 sCMOS camera and I was wondering if anybody has had any similar experiences, both in terms of feedback on what is expected from sCMOS hardware and what can be done to rectify any issues. Here's what we're seeing: Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're having three issues regarding hot and cold pixels. So far we have tried 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin Elmer), to double check the subtleties, but the results are quite similar On the first camera we saw a bright cluster of 4 pixels that are between 2 and 10x greater than the surrounding pixels across nearly all imaging conditions (not seen in the other three cameras). Are these bright pixel clusters something that others have seen much? This issue is seen in both Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used to cross check. I also see 'ghost' clusters that look similar but only seem to last for one frame... Again, is that something seen in sCMOS cameras in general? Salt: In images with low signal and longer exposure times we see speckled bight pixels using both Volocity and ExCap/HDImage, with an intensity approx twice that of the background signal. Exposure times ~secs; low signal <1000 counts per pixel. These speckled hot spots were seen with all sCMOS cameras to some degree. Pepper: In almost the inverse situation, where we have higher signals with lower exposure times were also seen on all sCMOS cameras (the first camera with the clear hotspot had very bad 'peppering' compared to the others). I know that the pco cameras used on the Deltavision OMX have reference images applied to reduce the affects of these artifacts, and I was wondering if that's something that can be applied post-acquisition. Any thoughts and/or feedback on what's normal for an sCMOS, and how to get the best out of these cameras would be much appreciated. sCMOS cameras certainly are fast and have a great signal to noise, so am I expecting too much from this generation of hardware? Thanks Neil A couple of images to compare salt'n'pepper pixels seen on three cameras are here: https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/view?usp=sharing https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/view?usp=sharing |
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In reply to this post by Andrew York
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Maybe I am missing the point here but won't the characterization paper that has been cited in this discussion- (but not yet read by me) produce at least a Dark Current image and hopefully a an image of the dark pixels on a lighter background. These two images could be used with some combination of multiplication and division to at least give a better looking image. I would have to spend more time than I have at the moment to consider the implications for quantitation, but whether you are trying to measure light intensity or count objects, background correction operation rarely cause problems, and usually help. If you can send me the two images cited and a sample image I woul be happy to play around with them and post my results back to you and/or the list. Chris Chris Tully Principal Consultant Image Incyte, LLC 240-475-9753 Image Incyte, LLC [hidden email] -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew York Sent: Wednesday, January 21, 2015 8:00 AM To: [hidden email] Subject: Re: sCMOS salt'n'pepper issues ***** 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. ***** Can you share raw data? Also, how are you doing your illumination? On Jan 20, 2015 11:19 PM, "Neil Anthony" <[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 all, I hope the > science/optics/hardware/software/students/post-docs/ > reviewers/managers/next-door-neighbors and FedEx are treating you well. > > We have a couple of issues with a recently installed Hamamatsu Flash > 4.0 sCMOS camera and I was wondering if anybody has had any similar > experiences, both in terms of feedback on what is expected from sCMOS > hardware and what can be done to rectify any issues. > > Here's what we're seeing: > Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a > spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're > having three issues regarding hot and cold pixels. So far we have > tried 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu > and Perkin Elmer), to double check the subtleties, but the results are > quite similar > > On the first camera we saw a bright cluster of 4 pixels that are > between 2 and 10x greater than the surrounding pixels across nearly > all imaging conditions (not seen in the other three cameras). Are > these bright pixel clusters something that others have seen much? This > issue is seen in both Volocity and ExCap/HDImage, but was not seen on > the test sCMOS camera used to cross check. I also see 'ghost' > clusters that look similar but only seem to last for one frame... > Again, is that something seen in sCMOS cameras in general? > > Salt: > In images with low signal and longer exposure times we see speckled > bight pixels using both Volocity and ExCap/HDImage, with an intensity > approx twice that of the background signal. Exposure times ~secs; low > signal <1000 counts per pixel. These speckled hot spots were seen > with all sCMOS cameras to some degree. > Pepper: > In almost the inverse situation, where we have higher signals with > lower exposure times were also seen on all sCMOS cameras (the first > camera with the clear hotspot had very bad 'peppering' compared to the others). > > I know that the pco cameras used on the Deltavision OMX have reference > images applied to reduce the affects of these artifacts, and I was > wondering if that's something that can be applied post-acquisition. > Any thoughts and/or feedback on what's normal for an sCMOS, and how to > get the best out of these cameras would be much appreciated. sCMOS > cameras certainly are fast and have a great signal to noise, so am I > expecting too much from this generation of hardware? > > Thanks > Neil > > > A couple of images to compare salt'n'pepper pixels seen on three > cameras are here: > https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/ > view?usp=sharing > https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/ > view?usp=sharing > |
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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. ***** Hello all, I think we need to remember that there are two possible sources of sCMOS pixels that record too much signal: high leakage (dark current or dark charge) and incorrect DC offset of the amplifier in the pixel. The former will show increasingly brighter pixels the longer the exposure. The latter will not. Use of an sCMOS with a disk-scanner implies to me probably longer exposures (where people often choose the EM-CCD) so maybe you need to need to perform the "pixel-by-pixel gain and offset" correction using the same exposure time that you plan to use for collecting data. Best, Jim Pawley >***** >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. >***** > >Maybe I am missing the point here but won't the characterization >paper that has been cited in this discussion- (but not yet read by >me) produce at least a Dark Current image and hopefully a an image >of the dark pixels on a lighter background. These two images could >be used with some combination of multiplication and division to at >least give a better looking image. I would have to spend more time >than I have at the moment to consider the implications for >quantitation, but whether you are trying to measure light intensity >or count objects, background correction operation rarely cause >problems, and usually help. > >If you can send me the two images cited and a sample image I woul be >happy to play around with them and post my results back to you >and/or the list. > >Chris > >Chris Tully >Principal Consultant >Image Incyte, LLC >240-475-9753 >Image Incyte, LLC >[hidden email] > > > >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of Andrew York >Sent: Wednesday, January 21, 2015 8:00 AM >To: [hidden email] >Subject: Re: sCMOS salt'n'pepper issues > >***** >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. >***** > >Can you share raw data? Also, how are you doing your illumination? >On Jan 20, 2015 11:19 PM, "Neil Anthony" <[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 all, I hope the >> science/optics/hardware/software/students/post-docs/ >> reviewers/managers/next-door-neighbors and FedEx are treating you well. >> >> We have a couple of issues with a recently installed Hamamatsu Flash >> 4.0 sCMOS camera and I was wondering if anybody has had any similar >> experiences, both in terms of feedback on what is expected from sCMOS >> hardware and what can be done to rectify any issues. >> >> Here's what we're seeing: >> Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a >> spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're >> having three issues regarding hot and cold pixels. So far we have >> tried 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu >> and Perkin Elmer), to double check the subtleties, but the results are >> quite similar >> >> On the first camera we saw a bright cluster of 4 pixels that are >> between 2 and 10x greater than the surrounding pixels across nearly >> all imaging conditions (not seen in the other three cameras). Are >> these bright pixel clusters something that others have seen much? This >> issue is seen in both Volocity and ExCap/HDImage, but was not seen on >> the test sCMOS camera used to cross check. I also see 'ghost' >> clusters that look similar but only seem to last for one frame... >> Again, is that something seen in sCMOS cameras in general? >> >> Salt: >> In images with low signal and longer exposure times we see speckled > > bight pixels using both Volocity and ExCap/HDImage, with an intensity >> approx twice that of the background signal. Exposure times ~secs; low >> signal <1000 counts per pixel. These speckled hot spots were seen >> with all sCMOS cameras to some degree. >> Pepper: >> In almost the inverse situation, where we have higher signals with >> lower exposure times were also seen on all sCMOS cameras (the first >> camera with the clear hotspot had very bad 'peppering' compared to >>the others). >> >> I know that the pco cameras used on the Deltavision OMX have reference >> images applied to reduce the affects of these artifacts, and I was >> wondering if that's something that can be applied post-acquisition. >> Any thoughts and/or feedback on what's normal for an sCMOS, and how to >> get the best out of these cameras would be much appreciated. sCMOS >> cameras certainly are fast and have a great signal to noise, so am I >> expecting too much from this generation of hardware? >> >> Thanks >> Neil >> >> >> A couple of images to compare salt'n'pepper pixels seen on three >> cameras are here: >> https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/ >> view?usp=sharing >> https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/ >> view?usp=sharing >> -- **************************************** James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, Phone 604-885-0840, email <[hidden email]> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146 |
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In reply to this post by Kyle Michael Douglass
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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 Kyle, thanks for all the details. Thanks for the link to that article; I'll get my teeth into that asap and post the results of how things go. Thanks Neil On 1/21/2015 3:25 AM, Kyle Douglass 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 Neil, > On 01/21/2015 04:24 AM, Neil Anthony wrote: >> >> On the first camera we saw a bright cluster of 4 pixels that are >> between 2 and 10x greater than the surrounding pixels across nearly >> all imaging conditions (not seen in the other three cameras). >> > > We have an Andor Zyla 4.2 and I've tested a separate Zyla and the > Flash 4.0 and I've never observed the clustering of hot spots. > >> Salt: >> In images with low signal and longer exposure times we see speckled >> bight pixels using both Volocity and ExCap/HDImage, with an intensity >> approx twice that of the background signal. > > These hot spots I believe come from the "anonymously" noisy chip-level > amplifiers. In an sCMOS camera, each pixel has its own amplifier and > it seems that it's nearly impossible to ensure that all ~4 million > pixels are defect free. In contrast, an EMCCD does not have > pixel-dependent noise properties because the amplification is not > pixel-dependent. Therefore, it seems like you have to accept hot spots > by thinking of them as manufacturing defects that occur in making a > large number of single pixels. > > We characterized our camera by measuring the pixel-dependent gain, > noise variance, and hot spot locations according to the procedure in > the supplement of this paper from the Bewersdorf lab: > http://www.ncbi.nlm.nih.gov/pubmed/23708387. We found that our > characterization matched the hot pixel map provided by Andor exactly, > so we know what the intensity statistics of those pixels are. > >> Pepper: >> In almost the inverse situation, where we have higher signals with >> lower exposure times were also seen on all sCMOS cameras (the first >> camera with the clear hotspot had very bad 'peppering' compared to >> the others). > > I'm not sure about this one. I haven't noticed the peppering. Is the > hot spot correction algorithm on in this case? >> >> I know that the pco cameras used on the Deltavision OMX have >> reference images applied to reduce the affects of these artifacts, >> and I was wondering if that's something that can be applied >> post-acquisition. > > We work in STORM/PALM microscopy and incorporate the hot spots into > our noise model when performing the localization analysis, much like > in the paper I cited above. We therefore do not use the manufactuer's > algorithm for hot pixel smoothing. > > For "normal" imaging, I think you either have to use the hot spot > correction algorithms to smooth over those pixels, or turn off the > algorithm and post-process the hot spots yourself. > > I hope this helps! > > Kyle > |
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In reply to this post by Neil Anthony
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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 Orla, thanks for offering to help with comparisons. A set of three test movies under dark conditions are available in a Volocity data set here: https://drive.google.com/file/d/0BzfJAGNfrgieQklEN1lnQnBKMmM/view?usp=sharing (use Fiji to open the mvd2 file if you don't have Volocity) The metadata should contain the details you need. For completeness, in response to your questions: 1. Exposure Time should be in the file name in the above 2. Frame rate (if time series taken) maximum possible 3. Trigger mode (external or internal etc) I assume internal as I don't have extra cables connected 4. Sensor Readout rate don't know 5. Sensor Temperature don't know 6. If you could provide a spec sheet for one of your cameras then I can correlate sensitivities to replicate their photon flux should be available from Hamamatsu 7. Are images single image or an image taken as part of a time series or an accumulated image? data above is time series without binning or accumulation etc. 8. Are you operating in Rolling Shutter or Global Reset Mode? I assume global, but not sure 9. Are you using Light Sheet Readout mode? no, not as far as I'm aware The control of the camera is set in Volocity developed by Perkin Elmer, and the majority of what you have requested above is not available to the user. I have not been able to catch a real big instance of ghost pixels in a non-sample image set (i.e. a situation where it cannot be a piece of biological schmutz jumping into and then out of the frame), but I have seen a handful during my control data sets. They almost look like they could be tiny pieces of dust jumping onto and off of the sensor, but I really can't tell if they're real or electronic. They are ~5 pixels wide and sometimes have a streak nature to them. A small version of what I'm seeing can be seen on frame 118 of the 100ms data set and frame 10 of the 2s data set. Once you've taken your data sets it would be great if you could share them with the list for comparison. Thanks Neil On 1/21/2015 7:04 AM, Orla Hanrahan wrote: > Dear Neil, > > We would like to run our Zyla 4.2 under the exact same operating conditions as you ran your Flash 4 to see if we get the same salt 'n' pepper effect. In order for us to replicate your images precisely we would need the following data from you : > > > 1. Raw images produced rather than screenshots (can you provide raw images?) > > 2. Details of the following for each image: > 1. Exposure Time > 2. Frame rate (if time series taken) > 3. Trigger mode (external or internal etc) > 4. Sensor Readout rate > 5. Sensor Temperature > 6. If you could provide a spec sheet for one of your cameras then I can correlate sensitivities to replicate their photon flux > 7. Are images single image or an image taken as part of a time series or an accumulated image? > 8. Are you operating in Rolling Shutter or Global Reset Mode? > 9. Are you using Light Sheet Readout mode? > > 3. Also an image showing the "ghost bright spot" would be great. > > With this info we could get a better idea of what is going on with the cameras you are currently using and would be able to tell you more accurately if we have similar issues. > > I look forward to hearing from you, > > With my very best wishes, > > Orla Hanrahan > > > ---------------------------------------------------------------------------------------- > Orla Hanrahan, PhD > Application Specialist, Life Science Imaging > > Tel: +44 (0)74 256 27982 > W: www.andor.com > Skype: orla.hanrahan1 > > > > > > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Neil Anthony > Sent: 21 January 2015 03:24 > To: [hidden email] > Subject: sCMOS salt'n'pepper issues > > ***** > 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 hope the > science/optics/hardware/software/students/post-docs/reviewers/managers/next-door-neighbors > and FedEx are treating you well. > > We have a couple of issues with a recently installed Hamamatsu Flash 4.0 sCMOS camera and I was wondering if anybody has had any similar experiences, both in terms of feedback on what is expected from sCMOS hardware and what can be done to rectify any issues. > > Here's what we're seeing: > Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're having three issues regarding hot and cold pixels. So far we have tried > 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin Elmer), to double check the subtleties, but the results are quite similar > > On the first camera we saw a bright cluster of 4 pixels that are between > 2 and 10x greater than the surrounding pixels across nearly all imaging conditions (not seen in the other three cameras). Are these bright pixel clusters something that others have seen much? This issue is seen in both Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used to cross check. I also see 'ghost' clusters that look similar but only seem to last for one frame... Again, is that something seen in sCMOS cameras in general? > > Salt: > In images with low signal and longer exposure times we see speckled bight pixels using both Volocity and ExCap/HDImage, with an intensity approx twice that of the background signal. Exposure times ~secs; low signal <1000 counts per pixel. These speckled hot spots were seen with all sCMOS cameras to some degree. > Pepper: > In almost the inverse situation, where we have higher signals with lower exposure times were also seen on all sCMOS cameras (the first camera with the clear hotspot had very bad 'peppering' compared to the others). > > I know that the pco cameras used on the Deltavision OMX have reference images applied to reduce the affects of these artifacts, and I was wondering if that's something that can be applied post-acquisition. Any thoughts and/or feedback on what's normal for an sCMOS, and how to get the best out of these cameras would be much appreciated. sCMOS cameras certainly are fast and have a great signal to noise, so am I expecting too much from this generation of hardware? > > Thanks > Neil > > > A couple of images to compare salt'n'pepper pixels seen on three cameras are here: > https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/view?usp=sharing > https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/view?usp=sharing > |
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In reply to this post by Andrew York
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Andrew, I have some dark images here: https://drive.google.com/file/d/0BzfJAGNfrgieQklEN1lnQnBKMmM/view?usp=sharing The illumination was provided by a 488 line of a gas laser through a (new) fiber optic to the early model Yokogawa spinning disk. Thanks Neil On 1/21/2015 7:59 AM, Andrew York 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. > ***** > > Can you share raw data? Also, how are you doing your illumination? > On Jan 20, 2015 11:19 PM, "Neil Anthony" <[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 all, I hope the science/optics/hardware/software/students/post-docs/ >> reviewers/managers/next-door-neighbors and FedEx are treating you well. >> >> We have a couple of issues with a recently installed Hamamatsu Flash 4.0 >> sCMOS camera and I was wondering if anybody has had any similar >> experiences, both in terms of feedback on what is expected from sCMOS >> hardware and what can be done to rectify any issues. >> >> Here's what we're seeing: >> Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a >> spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're >> having three issues regarding hot and cold pixels. So far we have tried 4 >> different sCMOS cameras (kindly supplied/tested by Hamamatsu and Perkin >> Elmer), to double check the subtleties, but the results are quite similar >> >> On the first camera we saw a bright cluster of 4 pixels that are between 2 >> and 10x greater than the surrounding pixels across nearly all imaging >> conditions (not seen in the other three cameras). Are these bright pixel >> clusters something that others have seen much? This issue is seen in both >> Volocity and ExCap/HDImage, but was not seen on the test sCMOS camera used >> to cross check. I also see 'ghost' clusters that look similar but only >> seem to last for one frame... Again, is that something seen in sCMOS >> cameras in general? >> >> Salt: >> In images with low signal and longer exposure times we see speckled bight >> pixels using both Volocity and ExCap/HDImage, with an intensity approx >> twice that of the background signal. Exposure times ~secs; low signal <1000 >> counts per pixel. These speckled hot spots were seen with all sCMOS >> cameras to some degree. >> Pepper: >> In almost the inverse situation, where we have higher signals with lower >> exposure times were also seen on all sCMOS cameras (the first camera with >> the clear hotspot had very bad 'peppering' compared to the others). >> >> I know that the pco cameras used on the Deltavision OMX have reference >> images applied to reduce the affects of these artifacts, and I was >> wondering if that's something that can be applied post-acquisition. Any >> thoughts and/or feedback on what's normal for an sCMOS, and how to get the >> best out of these cameras would be much appreciated. sCMOS cameras >> certainly are fast and have a great signal to noise, so am I expecting too >> much from this generation of hardware? >> >> Thanks >> Neil >> >> >> A couple of images to compare salt'n'pepper pixels seen on three cameras >> are here: >> https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/ >> view?usp=sharing >> https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/ >> view?usp=sharing >> |
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In reply to this post by Chris Tully-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. ***** Hi Chris, thanks for your feedback. A Volocity data set containing a few quick movies of a completely dark image at different exposures is available for download here: https://drive.google.com/file/d/0BzfJAGNfrgieQklEN1lnQnBKMmM/view?usp=sharing You can access the data by opening the mvd2 file using Fiji. (as a side note, I'm always surprised at how much I can zip the Volocity files; here from 566MB to 136MB!) Thanks Neil On 1/21/2015 9:10 AM, Chris Tully 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. > ***** > > Maybe I am missing the point here but won't the characterization paper that has been cited in this discussion- (but not yet read by me) produce at least a Dark Current image and hopefully a an image of the dark pixels on a lighter background. These two images could be used with some combination of multiplication and division to at least give a better looking image. I would have to spend more time than I have at the moment to consider the implications for quantitation, but whether you are trying to measure light intensity or count objects, background correction operation rarely cause problems, and usually help. > > If you can send me the two images cited and a sample image I woul be happy to play around with them and post my results back to you and/or the list. > > Chris > > Chris Tully > Principal Consultant > Image Incyte, LLC > 240-475-9753 > Image Incyte, LLC > [hidden email] > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andrew York > Sent: Wednesday, January 21, 2015 8:00 AM > To: [hidden email] > Subject: Re: sCMOS salt'n'pepper issues > > ***** > 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. > ***** > > Can you share raw data? Also, how are you doing your illumination? > On Jan 20, 2015 11:19 PM, "Neil Anthony" <[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 all, I hope the >> science/optics/hardware/software/students/post-docs/ >> reviewers/managers/next-door-neighbors and FedEx are treating you well. >> >> We have a couple of issues with a recently installed Hamamatsu Flash >> 4.0 sCMOS camera and I was wondering if anybody has had any similar >> experiences, both in terms of feedback on what is expected from sCMOS >> hardware and what can be done to rectify any issues. >> >> Here's what we're seeing: >> Using a Hamamatsu Flash 4.0 camera link sCMOS (firmware v3.00A) on a >> spinning disk and acquiring data using Perkin Elmer Volocity 6.3 we're >> having three issues regarding hot and cold pixels. So far we have >> tried 4 different sCMOS cameras (kindly supplied/tested by Hamamatsu >> and Perkin Elmer), to double check the subtleties, but the results are >> quite similar >> >> On the first camera we saw a bright cluster of 4 pixels that are >> between 2 and 10x greater than the surrounding pixels across nearly >> all imaging conditions (not seen in the other three cameras). Are >> these bright pixel clusters something that others have seen much? This >> issue is seen in both Volocity and ExCap/HDImage, but was not seen on >> the test sCMOS camera used to cross check. I also see 'ghost' >> clusters that look similar but only seem to last for one frame... >> Again, is that something seen in sCMOS cameras in general? >> >> Salt: >> In images with low signal and longer exposure times we see speckled >> bight pixels using both Volocity and ExCap/HDImage, with an intensity >> approx twice that of the background signal. Exposure times ~secs; low >> signal <1000 counts per pixel. These speckled hot spots were seen >> with all sCMOS cameras to some degree. >> Pepper: >> In almost the inverse situation, where we have higher signals with >> lower exposure times were also seen on all sCMOS cameras (the first >> camera with the clear hotspot had very bad 'peppering' compared to the others). >> >> I know that the pco cameras used on the Deltavision OMX have reference >> images applied to reduce the affects of these artifacts, and I was >> wondering if that's something that can be applied post-acquisition. >> Any thoughts and/or feedback on what's normal for an sCMOS, and how to >> get the best out of these cameras would be much appreciated. sCMOS >> cameras certainly are fast and have a great signal to noise, so am I >> expecting too much from this generation of hardware? >> >> Thanks >> Neil >> >> >> A couple of images to compare salt'n'pepper pixels seen on three >> cameras are here: >> https://drive.google.com/file/d/0BzfJAGNfrgieQ21abmVPbFBqY0k/ >> view?usp=sharing >> https://drive.google.com/file/d/0BzfJAGNfrgieVjViTWNoeExpdDQ/ >> view?usp=sharing >> |
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In reply to this post by Christian Soeller
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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 Christian, thanks for your reply and info. The camera was not an 'option' per se, but more an aftermarket upgrade we purchased from Perkin Elmer. They have been assisting us with testing out different instances of the Flash4.0 and we have the, on average, best one of the few we've tried. Thanks Neil On 1/21/2015 8:43 AM, Christian Soeller 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 Neil, > > We also have a Zyla 4.2 and I can mostly confirm Kyle's observation. >> Salt: >> In images with low signal and longer exposure times we see speckled >> bight pixels using both Volocity and ExCap/HDImage, with an intensity >> approx twice that of the background signal. Exposure times ~secs; low >> signal <1000 counts per pixel. These speckled hot spots were seen with >> all sCMOS cameras to some degree. >> >> >> > The salt (white pixels I presume) you see may be related to pixels with higher dark currents which may become troubling for long exposures. This makes those pixels not great for long exposures. > >> Pepper: >> In almost the inverse situation, where we have higher signals with lower >> exposure times were also seen on all sCMOS cameras (the first camera >> with the clear hotspot had very bad 'peppering' compared to the others). >> > The pepper, presumably very dark appearing pixels, could be due to at least two things. (1) a local low gain (gain is in principle pixel dependent) and (2) high read noise. > > A data post processing step is likely the best way to deal with some of these issues as are careful camera characterisation to guide that processing (see earlier reference to the Bewersdorf paper). Has anybody done this for confocal yet (rather than PALM/STORM)? In diffraction-limited imaging with suitable oversampling interpolation of missing data should be pretty doable. > > Do the manufacturers offer any assistance with this? I would have thought they should if they market these cameras as an option in these systems. > > Best, > Christian |
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