AOD v Resonant scanner
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi All, This would be for a MP application, so descanning is not an issue. I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off). Thanks in advance for your feedback. Best, Gary Gary Laevsky, Ph.D. Confocal Imaging Facility Manager Dept. of Molecular Biology Washington Rd. Princeton University Princeton, New Jersey, 08544-1014 (O) 609 258 5432 (C) 508 507 1310 |
 
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Smith, Benjamin E. |
Re: AOD v Resonant scanner
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hey Gary, We have the resonant scanner installed on our Leica SP8, and it certainly has some ups and downs: Pros: Video scan rates (8000 Hz) No discernible image distortion Zooms to 48x Pairs well with hybrid detectors (in line accumulate mode) Can be used with a pinhole aperture, resulting in higher axial resolution Cons: High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) No frequency control, you are stuck at the resonant frequency of the scanner Lots of shot noise in live scan mode (although all high speed scanners would have this). Another thing to consider is I've known AOTFs to burn out over time, but have not yet had a galvanometer burn out, so I would imagine a resonant scanner would last much longer than an AOTF that is rapidly cycling through a frequency regime. My guess is this, and the increased axial resolution, is why manufacturers stick with resonant scanners. You can also find a good discussion on the challenges of various high speed scan technologies here: http://www.microscopyu.com/articles/confocal/resonantscanning.html Hope this helps, Ben Smith ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Laevsky, Gary S. [[hidden email]] Sent: Friday, July 05, 2013 8:26 AM To: [hidden email] Subject: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi All, This would be for a MP application, so descanning is not an issue. I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off). Thanks in advance for your feedback. Best, Gary Gary Laevsky, Ph.D. Confocal Imaging Facility Manager Dept. of Molecular Biology Washington Rd. Princeton University Princeton, New Jersey, 08544-1014 (O) 609 258 5432 (C) 508 507 1310 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E. Sent: Saturday, 6 July 2013 12:14 AM To: [hidden email] Subject: Re: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hey Gary, We have the resonant scanner installed on our Leica SP8, and it certainly has some ups and downs: Pros: Video scan rates (8000 Hz) No discernible image distortion Zooms to 48x Pairs well with hybrid detectors (in line accumulate mode) Can be used with a pinhole aperture, resulting in higher axial resolution Cons: High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) No frequency control, you are stuck at the resonant frequency of the scanner Lots of shot noise in live scan mode (although all high speed scanners would have this). Another thing to consider is I've known AOTFs to burn out over time, but have not yet had a galvanometer burn out, so I would imagine a resonant scanner would last much longer than an AOTF that is rapidly cycling through a frequency regime. My guess is this, and the increased axial resolution, is why manufacturers stick with resonant scanners. You can also find a good discussion on the challenges of various high speed scan technologies here: http://www.microscopyu.com/articles/confocal/resonantscanning.html Hope this helps, Ben Smith ________________________________________ From: Confocal Microscopy List [[hidden email]] on behalf of Laevsky, Gary S. [[hidden email]] Sent: Friday, July 05, 2013 8:26 AM To: [hidden email] Subject: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi All, This would be for a MP application, so descanning is not an issue. I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off). Thanks in advance for your feedback. Best, Gary Gary Laevsky, Ph.D. Confocal Imaging Facility Manager Dept. of Molecular Biology Washington Rd. Princeton University Princeton, New Jersey, 08544-1014 (O) 609 258 5432 (C) 508 507 1310 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** > An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection. This shouldn't make a difference when using non-descanned detection with a single excitation line (e.g. 2P excitation), or am I missing something obvious? Cheers, Chris |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Sorry, I got into that message series a bit late and didn't see the earlier posts. You are quite right that an AOD should be really quite effective in 2-photon. I don't know of any current application but I agree that it looks really promising. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Wilms Sent: Saturday, 6 July 2013 6:41 PM To: [hidden email] Subject: Re: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** > An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection. This shouldn't make a difference when using non-descanned detection with a single excitation line (e.g. 2P excitation), or am I missing something obvious? Cheers, Chris |
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In reply to this post by Guy Cox-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 ***** Hi Guy, I do not see any slits in the AODs based LOTOS MPEF system published by Konnerth's lab: LOTOS-based two-photon calcium imaging of dendritic spines in vivo. <http://www.ncbi.nlm.nih.gov/pubmed/22976353> *Chen* X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL, *Konnerth* A. Nat Protoc. 2012 Oct;7(10):1818-29. doi: 10.1038/nprot.2012.106. Epub 2012 Sep 13. PMID: 22976353 Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization. The methods section does discuss a resonant scanner configuration. Hi Ben, You wrote, Cons: High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) No frequency control, you are stuck at the resonant frequency of the scanner Lots of shot noise in live scan mode (although all high speed scanners would have this). High pitched noise - well duh! The resonant scanner does work like a pitchfork. I recommend you buy earplugs or noise canceling headsets for everyone who works in the room. Error and shutdown: option A: complain to Leica that you have a bad instrument, and have them replace whatever is bad. option B: shut down the system every three hours. option C: "Don't do that!" - if you avoid lowest zoom, sounds like you can go longer. No frequency control: let me repeat: well duh! Lots of shot noise ... HyD's have about 2x quantum efficiency of the standard Leica PMTs. If you purchased standard PMTs, find the money to go HyD's. Also think about using denoising methods to get the most out of your data. For example, my suggestion that using the median for each pixel of an odd number of acquisitions (and others on te listserv have made alternative suggestions to this). Denoising techniques can have a huge impact on the usefulness of data - especially high speed, photon limited, image acquisitions. As a current example, Bewersdorf and colleagues recently published on how to fully characterize sCMOS sensor noise (which is different than PMT or HyD noise), to enable sCMOS to kick EMCCD's butt for single molecule localization: Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. <http://www.ncbi.nlm.nih.gov/pubmed/23708387> Huang F, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, *Bewersdorf J*. Nat Methods. 2013 Jul;10(7):653-8. doi: 10.1038/nmeth.2488. PMID: 23708387 Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells. http://www.nature.com/nmeth/journal/v10/n7/full/nmeth.2488.html#supplementary-information http://www.nature.com/nmeth/journal/v10/n7/extref/nmeth.2488-S1.pdf In particular, I recommend looking at Supplemental Figure 8 (pdf page 10) - supplemental files are free online. enjoy, George On 7/6/2013 12:40 AM, Guy Cox wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection. > > Guy > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E. > Sent: Saturday, 6 July 2013 12:14 AM > To: [hidden email] > Subject: Re: AOD v Resonant scanner > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hey Gary, > We have the resonant scanner installed on our Leica SP8, and it certainly has some ups and downs: > > Pros: > Video scan rates (8000 Hz) > No discernible image distortion > Zooms to 48x > Pairs well with hybrid detectors (in line accumulate mode) > Can be used with a pinhole aperture, resulting in higher axial resolution > > Cons: > High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) > The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. > We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) > No frequency control, you are stuck at the resonant frequency of the scanner > Lots of shot noise in live scan mode (although all high speed scanners would have this). > > Another thing to consider is I've known AOTFs to burn out over time, but have not yet had a galvanometer burn out, so I would imagine a resonant scanner would last much longer than an AOTF that is rapidly cycling through a frequency regime. My guess is this, and the increased axial resolution, is why manufacturers stick with resonant scanners. > > You can also find a good discussion on the challenges of various high speed scan technologies here: http://www.microscopyu.com/articles/confocal/resonantscanning.html > > Hope this helps, > Ben Smith > ________________________________________ > From: Confocal Microscopy List [[hidden email]] on behalf of Laevsky, Gary S. [[hidden email]] > Sent: Friday, July 05, 2013 8:26 AM > To: [hidden email] > Subject: AOD v Resonant scanner > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi All, > > This would be for a MP application, so descanning is not an issue. > > I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off). > > Thanks in advance for your feedback. > > > > > Best, > > Gary > > > > Gary Laevsky, Ph.D. > Confocal Imaging Facility Manager > Dept. of Molecular Biology > Washington Rd. > Princeton University > Princeton, New Jersey, 08544-1014 > (O) 609 258 5432 > (C) 508 507 1310 > > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** No, sorry, as I said in a previous post I came late into this thread and didn't realize that the proposal was for multi-photon. The only snag I can see is that the AOD will stretch the pulse quite a bit, but I guess you can fix that by pre-chirping. Guy -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara Sent: Sunday, 7 July 2013 12:57 AM To: [hidden email] Subject: Re: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Guy, I do not see any slits in the AODs based LOTOS MPEF system published by Konnerth's lab: LOTOS-based two-photon calcium imaging of dendritic spines in vivo. <http://www.ncbi.nlm.nih.gov/pubmed/22976353> *Chen* X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL, *Konnerth* A. Nat Protoc. 2012 Oct;7(10):1818-29. doi: 10.1038/nprot.2012.106. Epub 2012 Sep 13. PMID: 22976353 Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization. The methods section does discuss a resonant scanner configuration. Hi Ben, You wrote, Cons: High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) No frequency control, you are stuck at the resonant frequency of the scanner Lots of shot noise in live scan mode (although all high speed scanners would have this). High pitched noise - well duh! The resonant scanner does work like a pitchfork. I recommend you buy earplugs or noise canceling headsets for everyone who works in the room. Error and shutdown: option A: complain to Leica that you have a bad instrument, and have them replace whatever is bad. option B: shut down the system every three hours. option C: "Don't do that!" - if you avoid lowest zoom, sounds like you can go longer. No frequency control: let me repeat: well duh! Lots of shot noise ... HyD's have about 2x quantum efficiency of the standard Leica PMTs. If you purchased standard PMTs, find the money to go HyD's. Also think about using denoising methods to get the most out of your data. For example, my suggestion that using the median for each pixel of an odd number of acquisitions (and others on te listserv have made alternative suggestions to this). Denoising techniques can have a huge impact on the usefulness of data - especially high speed, photon limited, image acquisitions. As a current example, Bewersdorf and colleagues recently published on how to fully characterize sCMOS sensor noise (which is different than PMT or HyD noise), to enable sCMOS to kick EMCCD's butt for single molecule localization: Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. <http://www.ncbi.nlm.nih.gov/pubmed/23708387> Huang F, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, *Bewersdorf J*. Nat Methods. 2013 Jul;10(7):653-8. doi: 10.1038/nmeth.2488. PMID: 23708387 Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells. http://www.nature.com/nmeth/journal/v10/n7/full/nmeth.2488.html#supplementary-information http://www.nature.com/nmeth/journal/v10/n7/extref/nmeth.2488-S1.pdf In particular, I recommend looking at Supplemental Figure 8 (pdf page 10) - supplemental files are free online. enjoy, George On 7/6/2013 12:40 AM, Guy Cox wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > An AOD (as used in the old Noran systems) is highly chromatic and so cannot be used in fluorescence. AOD based fluorescence systems (Lasertek, Noran) have used the equivalent of slit, rather than spot, detection. > > > Guy > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Smith, Benjamin E. > Sent: Saturday, 6 July 2013 12:14 AM > To: [hidden email] > Subject: Re: AOD v Resonant scanner > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hey Gary, > We have the resonant scanner installed on our Leica SP8, and it certainly has some ups and downs: > > Pros: > Video scan rates (8000 Hz) > No discernible image distortion > Zooms to 48x > Pairs well with hybrid detectors (in line accumulate mode) > Can be used with a pinhole aperture, resulting in higher axial > resolution > > Cons: > High pitched frequency noise emitted form the scanner (which gets louder at lower zoom = larger amplitude) > The scanner lasts about 4-6 hours at its lowest zoom (1.25x) before giving an error and shutting down. > We couldn't ever get the bidirectional scan phase to line up perfectly (although we got it close) > No frequency control, you are stuck at the resonant frequency of the scanner > Lots of shot noise in live scan mode (although all high speed scanners would have this). > > Another thing to consider is I've known AOTFs to burn out over time, but have not yet had a galvanometer burn out, so I would imagine a resonant scanner would last much longer than an AOTF that is rapidly cycling through a frequency regime. My guess is this, and the increased axial resolution, is why manufacturers stick with resonant scanners. > > You can also find a good discussion on the challenges of various high > speed scan technologies here: > http://www.microscopyu.com/articles/confocal/resonantscanning.html > > Hope this helps, > Ben Smith > ________________________________________ > From: Confocal Microscopy List [[hidden email]] on > behalf of Laevsky, Gary S. [[hidden email]] > Sent: Friday, July 05, 2013 8:26 AM > To: [hidden email] > Subject: AOD v Resonant scanner > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi All, > > This would be for a MP application, so descanning is not an issue. > > I would think resonant galvos would be superior on the throughput side, whereas an AOD would win on speed and control (imagine, a trade-off). > > Thanks in advance for your feedback. > > > > > Best, > > Gary > > > > Gary Laevsky, Ph.D. > Confocal Imaging Facility Manager > Dept. of Molecular Biology > Washington Rd. > Princeton University > Princeton, New Jersey, 08544-1014 > (O) 609 258 5432 > (C) 508 507 1310 > > -- George McNamara, Ph.D. Single Cells Analyst L.J.N. Cooper Lab University of Texas M.D. Anderson Cancer Center Houston, TX 77054 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >No, sorry, as I said in a previous post I came late into this thread >and didn't realize that the proposal was for multi-photon. > >The only snag I can see is that the AOD will stretch the pulse quite >a bit, but I guess you can fix that by pre-chirping. > > Guy Hi all, I think that there may be another problem. As we used to say at the UBC Course, "If it ain't diffraction, it's statistics!" Guy has mentioned some of the diffraction effects of the AOD but I think that statistics may be even more important. With any single-beam scanning system, you will get 30x less signal/pixel during a (US) video scan than during a 1 second scan (assuming the same raster dimensions, beam power and degree of optical perfection). And even a one-second scan of "normal" (well-stained?) specimens often provides only 10-15 counts/pixel time from stained areas (assuming one of the hybrid PMTs with a GaAsP photocathode and no "excess noise"). With 2-photon, things are likely to be worse because even though the (squared) excitation volume is somewhat larger, the excitation is pulsed and at least 90% of the time you are not counting anything. And because of this low duty cycle, you are apt to be working at excitation levels a lot closer to singlet saturation than you should be (I assume that anyone interested in rapid changes must be interested in looking at living cells, not dying ones?) So, unless you can squeeze a LOT of dye molecules into the volumes the size of the focused spot (squared), your 2-photon, video rate images are likely to be very noisy indeed. This is why LaVision Biotech and many others trying to do 2-photon fast use multiple IR beams. But is does put a lot of power into (through?) the specimen. Cheers, Jim Pawley >-----Original Message----- >From: Confocal Microscopy List >[mailto:[hidden email]] On Behalf Of George >McNamara >Sent: Sunday, 7 July 2013 12:57 AM >To: [hidden email] >Subject: Re: AOD v Resonant scanner > >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hi Guy, > >I do not see any slits in the AODs based LOTOS MPEF system published >by Konnerth's lab: > > LOTOS-based two-photon calcium imaging of dendritic spines in vivo. > <http://www.ncbi.nlm.nih.gov/pubmed/22976353> > > *Chen* X, Leischner U, Varga Z, Jia H, Deca D, Rochefort NL, > *Konnerth* A. > > Nat Protoc. 2012 Oct;7(10):1818-29. doi: 10.1038/nprot.2012.106. > Epub 2012 Sep 13. > > PMID: > 22976353 > > Neurons in the mammalian brain receive thousands of synaptic inputs > on their dendrites. In many types of neurons, such as cortical > pyramidal neurons, excitatory synapses are formed on fine dendritic > protrusions called spines. Usually, an individual spine forms a > single synaptic contact with an afferent axon. In this protocol, we > describe a recently established experimental procedure for measuring > intracellular calcium signals from dendritic spines in cortical > neurons in vivo by using a combination of two-photon microscopy and > whole-cell patch-clamp recordings. We have used mice as an > experimental model system, but the protocol may be readily adapted > to other species. This method involves data acquisition at high > frame rates and low-excitation laser power, and is termed low-power > temporal oversampling (LOTOS). Because of its high sensitivity of > fluorescence detection and reduced phototoxicity, LOTOS allows for > prolonged and stable calcium imaging in vivo. Key aspects of the > protocol, which can be completed in 5-6 h, include the use of a > variant of high-speed two-photon imaging, refined surgery procedures > and optimized tissue stabilization. > > >The methods section does discuss a resonant scanner configuration. > >Hi Ben, > >You wrote, > > Cons: > High pitched frequency noise emitted form the scanner >(which gets louder at lower zoom = larger amplitude) > The scanner lasts about 4-6 hours at its lowest zoom >(1.25x) before giving an error and shutting down. > We couldn't ever get the bidirectional scan phase to line >up perfectly (although we got it close) > No frequency control, you are stuck at the resonant >frequency of the scanner > Lots of shot noise in live scan mode (although all high >speed scanners would have this). > > >High pitched noise - well duh! The resonant scanner does work like a >pitchfork. I recommend you buy earplugs or noise canceling headsets >for everyone who works in the room. > >Error and shutdown: > option A: complain to Leica that you have a bad instrument, and >have them replace whatever is bad. > option B: shut down the system every three hours. > option C: "Don't do that!" - if you avoid lowest zoom, sounds >like you can go longer. > >No frequency control: let me repeat: well duh! > >Lots of shot noise ... HyD's have about 2x quantum efficiency of the >standard Leica PMTs. If you purchased standard PMTs, find the money >to go HyD's. Also think about using denoising methods to get the >most out of your data. For example, my suggestion that using the >median for each pixel of an odd number of acquisitions (and others >on te listserv have made alternative suggestions to this). Denoising >techniques can have a huge impact on the usefulness of data - >especially high speed, photon limited, image acquisitions. As a >current example, Bewersdorf and colleagues recently published on how >to fully characterize sCMOS sensor noise (which is different than >PMT or HyD noise), to enable sCMOS to kick EMCCD's butt for single >molecule localization: > > Video-rate nanoscopy using sCMOS camera-specific single-molecule > localization algorithms. <http://www.ncbi.nlm.nih.gov/pubmed/23708387> > > Huang F, Hartwich TM, Rivera-Molina FE, Lin Y, Duim WC, Long JJ, > Uchil PD, Myers JR, Baird MA, Mothes W, Davidson MW, Toomre D, > *Bewersdorf J*. > > Nat Methods. 2013 Jul;10(7):653-8. doi: 10.1038/nmeth.2488. PMID: > > 23708387 > Newly developed scientific complementary metal-oxide semiconductor > (sCMOS) cameras have the potential to dramatically accelerate data > acquisition, enlarge the field of view and increase the effective > quantum efficiency in single-molecule switching nanoscopy. However, > sCMOS-intrinsic pixel-dependent readout noise substantially lowers > the localization precision and introduces localization artifacts. We > present algorithms that overcome these limitations and that provide > unbiased, precise localization of single molecules at the > theoretical limit. Using these in combination with a multi-emitter > fitting algorithm, we demonstrate single-molecule localization > super-resolution imaging at rates of up to 32 reconstructed images > per second in fixed and living cells. > >http://www.nature.com/nmeth/journal/v10/n7/full/nmeth.2488.html#supplementary-information >http://www.nature.com/nmeth/journal/v10/n7/extref/nmeth.2488-S1.pdf >In particular, I recommend looking at Supplemental Figure 8 (pdf page >10) - supplemental files are free online. > >enjoy, > >George > > > >On 7/6/2013 12:40 AM, Guy Cox wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> An AOD (as used in the old Noran systems) is highly chromatic and >>so cannot be used in fluorescence. AOD based fluorescence systems >>(Lasertek, Noran) have used the equivalent of slit, rather than >>spot, detection. >> >> >> Guy >> >> -----Original Message----- >> From: Confocal Microscopy List >>[mailto:[hidden email]] On Behalf Of Smith, >>Benjamin E. >> Sent: Saturday, 6 July 2013 12:14 AM >> To: [hidden email] >> Subject: Re: AOD v Resonant scanner > > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** >> >> Hey Gary, >> We have the resonant scanner installed on our Leica SP8, and >>it certainly has some ups and downs: >> >> Pros: >> Video scan rates (8000 Hz) >> No discernible image distortion >> Zooms to 48x >> Pairs well with hybrid detectors (in line accumulate mode) >> Can be used with a pinhole aperture, resulting in higher axial >> resolution >> >> Cons: >> High pitched frequency noise emitted form the scanner (which >>gets louder at lower zoom = larger amplitude) >> The scanner lasts about 4-6 hours at its lowest zoom (1.25x) >>before giving an error and shutting down. >> We couldn't ever get the bidirectional scan phase to line up >>perfectly (although we got it close) >> No frequency control, you are stuck at the resonant frequency >>of the scanner >> Lots of shot noise in live scan mode (although all high speed >>scanners would have this). >> >> Another thing to consider is I've known AOTFs to burn out over >>time, but have not yet had a galvanometer burn out, so I would >>imagine a resonant scanner would last much longer than an AOTF that >>is rapidly cycling through a frequency regime. My guess is this, >>and the increased axial resolution, is why manufacturers stick with >>resonant scanners. >> >> You can also find a good discussion on the challenges of various high >> speed scan technologies here: >> http://www.microscopyu.com/articles/confocal/resonantscanning.html >> >> Hope this helps, >> Ben Smith >> ________________________________________ >> From: Confocal Microscopy List [[hidden email]] on >> behalf of Laevsky, Gary S. [[hidden email]] >> Sent: Friday, July 05, 2013 8:26 AM >> To: [hidden email] >> Subject: AOD v Resonant scanner >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi All, >> >> This would be for a MP application, so descanning is not an issue. >> >> I would think resonant galvos would be superior on the throughput >>side, whereas an AOD would win on speed and control (imagine, a >>trade-off). >> >> Thanks in advance for your feedback. >> >> >> >> >> Best, >> >> Gary >> >> >> >> Gary Laevsky, Ph.D. >> Confocal Imaging Facility Manager >> Dept. of Molecular Biology >> Washington Rd. >> Princeton University >> Princeton, New Jersey, 08544-1014 >> (O) 609 258 5432 >> (C) 508 507 1310 >> >> > > >-- > > > >George McNamara, Ph.D. >Single Cells Analyst >L.J.N. Cooper Lab >University of Texas M.D. Anderson Cancer Center Houston, TX 77054 -- 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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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** > With 2-photon, things are likely to be worse because even though the (squared) excitation volume is somewhat larger, the excitation is pulsed and at least 90% of the time you are not counting anything. And because of this low duty cycle, you are apt to be working at excitation levels a lot closer to singlet saturation than you should be (I assume that anyone interested in rapid changes must be interested in looking at living cells, not dying ones?) You can use the low duty cycle to your advantage, though: Using a low number of excitation pulses per pixel by scanning very quickly (up to kHz frame rates) while using low excitation powers is the idea behind 2-photon LOTOS microscopy as used by Chen et al. from Arthur Konnerth's group (protocol paper: http://www.nature.com/nprot/journal/v7/n10/abs/nprot.2012.106.html). This would require AODs rather than resonance scanning to do it properly. > This is why LaVision Biotech and many others trying to do 2-photon fast use multiple IR beams. But is does put a lot of power into (through?) the specimen. Isn't the alternative of "High repetition rates with low power pulses" as implemented by Ji, Magee and Betzig (http://www.nature.com/nmeth/journal/v5/n2/full/nmeth.1175.html) potentially more promising (and easier to do)? This puts less power into the specimen while still giving the user many excitation pulses per pixel while scanning at high frame rates. I would assume that this approach combined with LOTOS might give users the best SNR at high speeds. In which case I would put my money into AODs… But it is possible that I am overlooking something, so please to correct me if I am! Cheers, Chris |
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi All, I currently have a Prairie system which is a point scanning system with an AOD that works at a variety of wavelengths. I believe Prairie may have adopted technology from earlier Noran systems as Prairie also offered a Noran-like slit-scanner at one point (not sure if they still do). I agree with Dr Pawley that the signal is "noisy" when using the system in AOD mode but this does not invalidate the methodology. I think that the AOD is best used for data acquisition and not for pretty images. Pre-chirping and GVD have been discussed at length on the list and elsewhere, and to summarize; yes pre-chirping would probably help this technique. I currently do not pre-chirp the beam in our system. Cheers, Brian D Armstrong PhD Associate Research Professor Director, Light Microscopy Core Beckman Research Institute City of Hope Dept of Neuroscience 1450 E Duarte Rd Duarte, CA 91010 626-256-4673 x62872 http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christian Wilms Sent: Monday, July 08, 2013 7:25 AM To: [hidden email] Subject: Re: AOD v Resonant scanner ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** > With 2-photon, things are likely to be worse because even though the (squared) excitation volume is somewhat larger, the excitation is pulsed and at least 90% of the time you are not counting anything. And because of this low duty cycle, you are apt to be working at excitation levels a lot closer to singlet saturation than you should be (I assume that anyone interested in rapid changes must be interested in looking at living cells, not dying ones?) You can use the low duty cycle to your advantage, though: Using a low number of excitation pulses per pixel by scanning very quickly (up to kHz frame rates) while using low excitation powers is the idea behind 2-photon LOTOS microscopy as used by Chen et al. from Arthur Konnerth's group (protocol paper: http://www.nature.com/nprot/journal/v7/n10/abs/nprot.2012.106.html). This would require AODs rather than resonance scanning to do it properly. > This is why LaVision Biotech and many others trying to do 2-photon fast use multiple IR beams. But is does put a lot of power into (through?) the specimen. Isn't the alternative of "High repetition rates with low power pulses" as implemented by Ji, Magee and Betzig (http://www.nature.com/nmeth/journal/v5/n2/full/nmeth.1175.html) potentially more promising (and easier to do)? This puts less power into the specimen while still giving the user many excitation pulses per pixel while scanning at high frame rates. I would assume that this approach combined with LOTOS might give users the best SNR at high speeds. In which case I would put my money into AODs... But it is possible that I am overlooking something, so please to correct me if I am! Cheers, Chris --------------------------------------------------------------------- *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p) --------------------------------------------------------------------- |
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Periasamy, Ammasi (ap3t) |
Job-Postdoctoral Research Associate
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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 ***** Dear All Please do not send your Biosketch to me. To apply, please submit a candidate profile on-line through Jobs@UVA (https://jobs.virginia.edu) Please read the advertisement listed below: The W.M. Keck Center for Cellular Imaging (KCCI), Department of Biology at the University of Virginia invites applications for a post-doctoral Research Associate position. The incumbent will be developing a new optical microscopy and spectroscopy technique to investigate protein-protein interaction using endogenous and exogenous fluorophore in living cells or tissues. The Research Associate will be responsible for administrating and training the center users on various microscopy systems including wide-field, confocal, multiphoton, TIRF, polarization, anisotropy, Second harmonic generation, high content screening system and lifetime imaging microscopy. Also, participate in the operation and training of international participants attending the annual workshop on FRET microscopy. The research associate should have an interest/ability in interacting with multidisciplinary investigators involved in the KCCI. A Ph.D. in physics, or Biophotonics, or biomedical engineering or a related discipline by appointment start date is required. Candidates with a background on various light microscopy systems and particular interest in design and development of a new optical instrumentation are preferred. Candidates must have effective oral and written communication skills. To apply, please submit a candidate profile on-line through Jobs@UVA (https://jobs.virginia.edu) and attach the following: a cover letter, curriculum vitae (CV), and the contact information for three references; Search on posting number 0612771. Review of applications will begin September 7, 2013; however, the position will remain open until filled. Questions regarding this position should be directed to: Dr. Ammasi Periasamy 434-243-7602 [hidden email] Questions regarding the Candidate Profile process or Jobs@UVA should be directed to: Richard Haverstrom 434-982-5615 [hidden email] The College of A&S and the University of Virginia welcome applications from women, minorities, veterans and persons with disabilities; we seek to build a culturally diverse, intellectual environment and are committed to a policy of equal employment opportunity and to the principles of affirmative action in accordance with state and federal laws. Dr. Ammasi Periasamy Professor & Center Director W.M. Keck Center for Cellular Imaging (KCCI) (University of Virginia Imaging Center) Mail or FedEx or UPS: Keck Center for Cellular Imaging University of Virginia Biology, Gilmer Hall, 485 McCormick Rd. Charlottesville, VA 22904, USA Office Location: Physical and Life Sciences Building (PLSB-B005), White Head Rd., Voice: 434-243-7602 (Office); 982-4869 (lab) Fax:434-982-5210; Email:[hidden email] http://www.kcci.virginia.edu/contact/peri.php ************************ 13th Annual Workshop on FRET & FLIM Microscopy, March 10-15, 2014 http://www.kcci.virginia.edu/workshop/workshop2014/index.php ************************* |
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Marco Marcello |
Job-Postdoctoral Research Associate- Imaging scientist
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*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear All Here at the Liverpool Centre for Cell Imaging (CCI) we are looking for an imaging scientist to help in the analysis of advanced microscopy experiments (FRAP, FLIM-FRET, RICS, FCS) and to lead the development of a novel super-resolution techniques (SOFI). You should have a PhD in Computer Science, Physics, Biology or related subject with expertise in biological image processing and computer programming. The post is available until 30 November 2016. To apply, please download an application form here: https://www.liv.ac.uk/working/job_vacancies/research/r-582859 Questions regarding this position should be directed to: Dr. Marco Marcello [hidden email] http://pcwww.liv.ac.uk/~cci/ Thanks, Marco Dr. Marco Marcello Light Microscopy Facility Manager Institute of Integrative Biology University of Liverpool L69 7ZB Liverpool UK http://pcwww.liv.ac.uk/~cci/ |
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