scan speed
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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 can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh |
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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 Timothy, In general, what you have said would be true for a constant laser power. You should consider that for bleaching what often matters most (excluding exotic imaging media as in STORM) is the product of intensity and duration- thus a short scan time *or* a low laser power are superficially similarly protective. However if your aim is to maximize SNR, a short dwell time increases the relative contribution of dark noise from the detector. Moreover if your sample is alive, phototoxicity increases (usually in a dramatically nonlinear fashion) with illumination intensity. In general my advice to students is to figure out how fast you need to go to comfortably address a particular question with your experiment, then back off on the laser as much as possible to protect your sample. -Pat Robison Sent from Mail for Windows 10 From: Feinstein, Timothy N Sent: Friday, October 18, 2019 10:16 AM To: [hidden email] Subject: scan speed ***** 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 can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh |
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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. ***** Regarding scan speed: Practically, how we train people for fixed and stained material is different than for live material. Consistent with the demand for minimal training (cost, getting data fast, not valuing intellectual development, disregarding QC), people who have fixed stained material tend to use slower scan speeds (or more averaging) and higher laser intensities to reduce noise. (They also tend to saturate their images the moment we leave the training session even though avoiding saturation is a point we reiterate repeatedly with demonstrations on how to recognize it and how to avoid it.) And there are some users who are oblivious to noise; perhaps some practical arts courses should be required. Some users opt for real knowledge of the confocal, but for most we tend to park them with 1024 X 1024 image size (Zeiss 700, 710, 780, and 880) at a zoom of 1X to 1.3X with pinhole at 1 Airy unit. Most of the new probes are very stable and combined with sensitive detectors, bleaching is not really a problem for the simple picture takers including those with Z series. (Airyscan is a different story...) Live work is different. We work closely with these researchers to balance field size, pinhole, scan speed, laser intensities, gain, averaging, # of intervals in Z and time to really needed to see biological event of interest, etc. Most of these users learn more about how the confocal works (and microscopes in general), but not all of them. This is more typical and extend it to at least half of our interactions. This morning on the way to work I got a frantic email about how the laser doesn't work on the Leica SP5. It will not turn on no matter what, the user restarted the instrument a few times. I go to check on it and the lasers on the SP8 (it's not a SP5) turn on fine. So I call the person in and ask him to put a slide on the scope. He cannot see anything by eye because the fluorescent lamp isn't sending light to the scope. The problem is that the user before him turned the attenuation knob on the front of metal halide lamp to the closed position (probably it was someone who understood the light path well and was protecting their sample). As we talked, he didn't turn off the light that was now blasting his sample. I suggested he close the shutter while we talked so he wouldn't bleach his sample. Yes, our training does include a description of the light sources and light paths. I'll stop here. Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 [hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/ Voice direct only, no text or messages: 1-914-309-3270 and 1-646-501-0567 ________________________________ From: Confocal Microscopy List <[hidden email]> on behalf of Pat Robison <[hidden email]> Sent: Friday, October 18, 2019 10:52:01 AM To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=Ys4UEUOSb4C0DCAP55y8SXGEiq02HPl8_wslmyx2WD4&s=YGtNu3hES7W6CCQllgO9Sh0lwRQGuzvf9ziQtweHr8A&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=Ys4UEUOSb4C0DCAP55y8SXGEiq02HPl8_wslmyx2WD4&s=zHTTgn5-FYrEIaDHucLaAjZzXxoHDFtqAXEpjQnyFdk&e= and include the link in your posting. ***** Hi Timothy, In general, what you have said would be true for a constant laser power. You should consider that for bleaching what often matters most (excluding exotic imaging media as in STORM) is the product of intensity and duration- thus a short scan time *or* a low laser power are superficially similarly protective. However if your aim is to maximize SNR, a short dwell time increases the relative contribution of dark noise from the detector. Moreover if your sample is alive, phototoxicity increases (usually in a dramatically nonlinear fashion) with illumination intensity. In general my advice to students is to figure out how fast you need to go to comfortably address a particular question with your experiment, then back off on the laser as much as possible to protect your sample. -Pat Robison Sent from Mail for Windows 10 From: Feinstein, Timothy N Sent: Friday, October 18, 2019 10:16 AM To: [hidden email] Subject: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=Ys4UEUOSb4C0DCAP55y8SXGEiq02HPl8_wslmyx2WD4&s=YGtNu3hES7W6CCQllgO9Sh0lwRQGuzvf9ziQtweHr8A&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=hUBj2D5n6oKThx2L01qn8IORZb5f-ruLVXPmQ1zQNnM&m=Ys4UEUOSb4C0DCAP55y8SXGEiq02HPl8_wslmyx2WD4&s=zHTTgn5-FYrEIaDHucLaAjZzXxoHDFtqAXEpjQnyFdk&e= and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh |
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In reply to this post by Tim Feinstein
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email] Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** 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 can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. (LCP301) ------------------------------------------------------------ |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** I also always train to scan at max. speed, but in my head it’s mostly to avoid quenching (bleaching too of course). -Esteban On Fri, Oct 18, 2019 at 10:22 AM Brian Armstrong <[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, well I have always thought that the longer the pixel dwell time the > more accurate the representation the image is of the sample. However, there > must be an optimal scan speed that relates to Nyquist sampling (Nyquist > indeed first theorized about temporal sampling). Perhaps modern electronics > perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that > sampling speed just isn't a factor in modern Confocal imaging as far as the > electronics are concerned. That being said there must be a "sweet spot" for > temporal sampling although it isn't really discussed much. I default to > scan speed of 1.02usec/pixel. I would be happy to hear how and why I am > wrong. > > As far as the number of Prinicipal Investigators at my institution that > would be willing to pay for training of 16 hours on a Confocal (mind you, > our time is free, they simply pay for the instrument time) is approximately > zero. Moreover, in my opinion there isn't a sound reason why each user > needs to have the expertise of a microscope guru. That would negate one key > reason why the core model is valid (the other being instrument cost). > Furthermore, teaching someone the intricacies of z-stack acquisition for 8 > hours who isn't performing z-stacks seems unnecessary (for example). > > Cheers, > > Brian Armstrong PhD > Associate Research Professor > Developmental and Stem Cell Biology > Diabetes and Metabolic Diseases > Director, Light Microscopy Core > Beckman Research Institute, City of Hope > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Feinstein, Timothy N > Sent: Friday, October 18, 2019 7:17 AM > To: [hidden email] > Subject: scan speed > > [Attention: This email came from an external source. Do not open > attachments or click on links from unknown senders or unexpected emails.] > > > > > > ***** > 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 can't understate how helpful this discussion about training and best > practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like > some others I encourage users to just set the fastest speed and leave it > there. Leica SP8s constrain the field of view with faster speed, so I > suggest SP8 users choose 700 Hz as a good balance between speed and field > of view. My reasoning comes strictly from my own testing - I found that > you pay a lot more in bleaching when you try to get a brighter signal by > slowing the scan than if you just turn up the laser power. Since the > slowest speeds drastically increase bleaching, I use them for a bleaching / > photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and > bleaching has to do with longer dwell time increasing the chance of > exciting fluorophores to the triplet state. Hence why resonant scan plus > line averaging is gentler on a live sample than a galvo scan of comparable > signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > > > ------------------------------------------------------------ > -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. > (LCP301) > ------------------------------------------------------------ > |
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In reply to this post by Tim Feinstein
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi all, I teach our users what scan speed means and why you sometimes use a fast speed and sometimes slow. I don't have a hard and fast rule but do have recommendations depending on the system for settings that work well for most fixed samples. For live cell/tissue imaging, there are often compromises to be made, i.e. scan speed usually higher/faster and no averaging and low laser power particularly if we need high temporal resolution and we will also sometimes open up the pinhole as I mentioned in an earlier post. I think it's important for the users to understand what scan speed means though so that they can make their own decisions. The level of noise in the image is the main thing that determines what scan speed and averaging we use. If the voltage/master gain is low, then we can go fast and don't have to use averaging. However, if the voltage/master gain is high and we have fixed samples with good quality mountant, then we have choices to make, i.e. do we increase laser power, slow scan speed down, use averaging or what is most usual, a combination of all three? If users are acquiring z stacks, then scan speed is very important as the time for acquisition is then multiplied up. It's at this time that they might decide to skimp on sampling in z (acquire fewer slices) so understanding what they can do to enable a faster scan speed to be used with good quality images is helpful as long as they are also mindful of photobleaching. I stress to the users that they should always check the quality of their image by looking at 100% of the pixels. If they don't do this and have the image window set to display the entire image in a smaller space, they will not see the noise and will be disappointed later. For labelling of punctate structures, it's even more important not to have excess noise present. Kind regards, Jacqui -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Saturday, 19 October 2019 3:17 a.m. To: [hidden email] Subject: scan speed ***** 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 can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh |
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In reply to this post by Armstrong, Brian
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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. ***** Our rationale for scan speed, averaging... is: - what is your scientific question: i.e. how will you analyse the images and what is the smallest distance that you need to measure in the sample (distance between objects, not size of objects)? - First choice is widefield, second spinning disk confocal, third single point confocal. And light sheet for large samples whenever we can avoid sectioning. - on a single point confocal, set everything to go as fast as possible (large pixels, fastest scan speed, no averaging). - does the acquired image allow you to answer your scientific question (measure the distance defined above)? - if yes, keep it that way. Set the machine to image automatically lots of sample and go and do something else while it works for you. 😊 - If not, if the pixel size is a problem, check if the chosen objective delivers a resolution that answers the scientific question. Check what the Nyquist pixel size is. Make a decision. Then check the noise by scanning continuously (which we normally never do. We only use snap) and seeing what changes from one image to the next: that is random noise. Random noise can be helped by lowering the gain (increasing lasers if possible), averaging and/or allowing more photons to the detector (more laser if possible, slower scan speed if possible, maybe using a lower magnification with high NA) - If the image is meant to be printed in a paper instead of being for data acquisition, just blast the thing with small pixels, high averaging, high laser... Just get 1 lovely image that way that can be magnified in case you get the journal cover then go back to acquiring 'real' data where all settings are chosen to reliably answer the scientific question. Any comments on our approach are very welcome. 😊 Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Brian Armstrong Sent: 18 October 2019 19:22 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C0481d41463204c15201f08d753efc2cc%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637070161556740296&sdata=qwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C0481d41463204c15201f08d753efc2cc%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637070161556740296&sdata=KgbaDOG2MLiJQIA%2BL8uH4LGV79BhrfQk5e562tfkG3g%3D&reserved=0 and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email] Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C0481d41463204c15201f08d753efc2cc%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637070161556740296&sdata=qwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C0481d41463204c15201f08d753efc2cc%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637070161556740296&sdata=KgbaDOG2MLiJQIA%2BL8uH4LGV79BhrfQk5e562tfkG3g%3D&reserved=0 and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. 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In reply to this post by Tim Feinstein
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Timothy, the spinning-disk people say that the critical point about bleaching is maximum peak power, and I tend to believe it. I think what you describe could be true for cw lasers, but maybe not for pulsed lasers, where the fluors have more time to drop back to ground state before the next excitation hits. In any case important would be laser power. But I admit I did not thoroughly test this. And should it really make a difference whether the pixel dwell time is 0.5 or 1 or 2 µs? In both cases there is ample time to go to T-states. Resonant scanning with times <50 ns may be different. Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I encourage our users to test for bleaching by recording the same image (stack) twice and check for intensity decrease in the second stack. If there is none, time can be save by higher laser power (up to the point of saturation). Fortunately not many of our users use FITC or APC, and modern fluors are pretty stable. Steffen Am 18.10.2019 um 16:16 schrieb Feinstein, Timothy N: > ***** > 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 can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany http://www.bioimaging.bmc.med.uni-muenchen.de |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Interesting discussion 😊 During our yearly 1-month course (Thanks Steffen for the clarification! It makes sense!), we have a workshop called The art of bleaching. We prepare non-confluent flat cells on coverslips and mounted on a slide. The cells are stained with a tubulin antibody and we use a secondary with FITC or Alexa Fluor 488. We mount the cells in either PBS (with nail polish) or Prolong Diamond. Then we go with our 4 samples to 4 different imaging modalities (4 student groups in parallel): widefield, spinning disk, single point confocal with galvo scanner, single point confocal with resonant scanner (averaging 4x). We first image continuously (i.e. we record a time lapse with no delay between images) for 3 min, then we immediately start a 2 min timer and when it beeps we acquire 1 more image (at the same exact position). Finally we remove the background everywhere and we compute the following: - intensity at start - intensity after 3 min continuous image or time to reach 50% bleaching - intensity of the final image after 2 min in the dark We set the acquisition parameters to get similar image qualities on all systems. Very fun! The results are: - with a spinning disk or a widefield system, we are not nearly closed to 50% bleaching after 3 min of continuous imaging - with a single point confocal, galvo or resonant, we reach 50% bleaching before the end of the 3 min - the 2 fluorophores behave differently and the mounting medium has a strong influence: FITC in prolong diamond bleaches much slower than AF488 in diamond. It is the opposite for PBS. - the intensity of FITC in PBS is brighter after 2 min in the dark Conclusions: - single point confocals bleach tons. This means that live cells will suffer a lot more than on a widefield/spinning disk - Never scanning continuously while setting the parameters. Take one image, assess, change the settings, take another image... a quick continuous scanning can be used to refocus. Original focusing should be done with bright field. - different fluorophores, even with the same colour, have different photophysics - the mounting medium is important to prevent bleaching. Choose a mounting medium that works for your fluorophore. - when the sample fluorescence goes down, the fluorophores could be bleaching or they could go to the dark state (from which they recover with time) Nothing surprising. All this is expected but demonstrating it during the course makes it go home and it is a lot of fun! One could indefinitely demonstrate all sorts of stuff with this set up, including laser power vs scan speed, line vs frame averaging... Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: 21 October 2019 10:37 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=OCCLfau9LMQ8l%2B8HFXjh1oALjhho6jIWk%2FtXasnAYzI%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=q8lsnHzgrCCyWirw6uvwKpgBZfqwRcNEC%2Fae8OOhf1s%3D&reserved=0 and include the link in your posting. ***** Hi Timothy, the spinning-disk people say that the critical point about bleaching is maximum peak power, and I tend to believe it. I think what you describe could be true for cw lasers, but maybe not for pulsed lasers, where the fluors have more time to drop back to ground state before the next excitation hits. In any case important would be laser power. But I admit I did not thoroughly test this. And should it really make a difference whether the pixel dwell time is 0.5 or 1 or 2 µs? In both cases there is ample time to go to T-states. Resonant scanning with times <50 ns may be different. Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I encourage our users to test for bleaching by recording the same image (stack) twice and check for intensity decrease in the second stack. If there is none, time can be save by higher laser power (up to the point of saturation). Fortunately not many of our users use FITC or APC, and modern fluors are pretty stable. Steffen Am 18.10.2019 um 16:16 schrieb Feinstein, Timothy N: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cs > ylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1 > cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=OCCLfa > u9LMQ8l%2B8HFXjh1oALjhho6jIWk%2FtXasnAYzI%3D&reserved=0 > Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217807741&sdata=3YlNePp6l0Gd28HO9T3eZsfLEIAR5sqrGJ8IX7Lbdac%3D&reserved=0 and include the link in your posting. > ***** > > Hi all, > > I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217807741&sdata=36xvdJY5%2Fh8%2B8e7PKdYs8GDBv4Qriv99QEIPBvQtQgc%3D&reserved=0 När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>. |
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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. ***** Hej Sylvie, A couple of thoughts 1) "We set the acquisition parameters to get similar image qualities on all systems" Same resolution, photon counts and image acquisition rates ? difficult. 2) most confocals and multiphoton microscopes are point scanning, so a continuous scan of a large area and a small area will produce an apparent differences in the bleaching rate. 3) bleaching per se is not the real issue, instead we perhaps should consider how many photons are detected for a given amount of photobleaching - how efficient are different microscopes at obtaining photons. A more extreme case is that for a fixed specimen and depending on our question, it doesn’t matter if the sample is finally fully photobleached - since this actually means we have obtained the best possible image - all the available photons have been captured and my slide has 5 000 more cells I can image. 4) Scan speed - is it for a given amount of photobleaching, better to use one slow scan or to average/integrate several faster scans adding up to the same scan time. The multiple fast scans involve generating more pixels, with presumably some additional noise, but since the scan is faster the local excitation power is lower and some systems, live tissue and mountants with antifade agents, should be cope better as their protection will not be overwhelmed by a higher peak power. So faster scanning should be better. I am not arguing that confocals do not produce bleaching, only that our measures of bleaching and the importance of bleaching to our question can be highly variable. Jeremy Adler -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader Sent: den 21 oktober 2019 11:35 To: [hidden email] Subject: Re: scan speed ***** 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. ***** Interesting discussion 😊 During our yearly 1-month course (Thanks Steffen for the clarification! It makes sense!), we have a workshop called The art of bleaching. We prepare non-confluent flat cells on coverslips and mounted on a slide. The cells are stained with a tubulin antibody and we use a secondary with FITC or Alexa Fluor 488. We mount the cells in either PBS (with nail polish) or Prolong Diamond. Then we go with our 4 samples to 4 different imaging modalities (4 student groups in parallel): widefield, spinning disk, single point confocal with galvo scanner, single point confocal with resonant scanner (averaging 4x). We first image continuously (i.e. we record a time lapse with no delay between images) for 3 min, then we immediately start a 2 min timer and when it beeps we acquire 1 more image (at the same exact position). Finally we remove the background everywhere and we compute the following: - intensity at start - intensity after 3 min continuous image or time to reach 50% bleaching - intensity of the final image after 2 min in the dark We set the acquisition parameters to get similar image qualities on all systems. Very fun! The results are: - with a spinning disk or a widefield system, we are not nearly closed to 50% bleaching after 3 min of continuous imaging - with a single point confocal, galvo or resonant, we reach 50% bleaching before the end of the 3 min - the 2 fluorophores behave differently and the mounting medium has a strong influence: FITC in prolong diamond bleaches much slower than AF488 in diamond. It is the opposite for PBS. - the intensity of FITC in PBS is brighter after 2 min in the dark Conclusions: - single point confocals bleach tons. This means that live cells will suffer a lot more than on a widefield/spinning disk - Never scanning continuously while setting the parameters. Take one image, assess, change the settings, take another image... a quick continuous scanning can be used to refocus. Original focusing should be done with bright field. - different fluorophores, even with the same colour, have different photophysics - the mounting medium is important to prevent bleaching. Choose a mounting medium that works for your fluorophore. - when the sample fluorescence goes down, the fluorophores could be bleaching or they could go to the dark state (from which they recover with time) Nothing surprising. All this is expected but demonstrating it during the course makes it go home and it is a lot of fun! One could indefinitely demonstrate all sorts of stuff with this set up, including laser power vs scan speed, line vs frame averaging... Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: 21 October 2019 10:37 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=OCCLfau9LMQ8l%2B8HFXjh1oALjhho6jIWk%2FtXasnAYzI%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=q8lsnHzgrCCyWirw6uvwKpgBZfqwRcNEC%2Fae8OOhf1s%3D&reserved=0 and include the link in your posting. ***** Hi Timothy, the spinning-disk people say that the critical point about bleaching is maximum peak power, and I tend to believe it. I think what you describe could be true for cw lasers, but maybe not for pulsed lasers, where the fluors have more time to drop back to ground state before the next excitation hits. In any case important would be laser power. But I admit I did not thoroughly test this. And should it really make a difference whether the pixel dwell time is 0.5 or 1 or 2 µs? In both cases there is ample time to go to T-states. Resonant scanning with times <50 ns may be different. Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I encourage our users to test for bleaching by recording the same image (stack) twice and check for intensity decrease in the second stack. If there is none, time can be save by higher laser power (up to the point of saturation). Fortunately not many of our users use FITC or APC, and modern fluors are pretty stable. Steffen Am 18.10.2019 um 16:16 schrieb Feinstein, Timothy N: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cs > ylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1 > cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=OCCLfa > u9LMQ8l%2B8HFXjh1oALjhho6jIWk%2FtXasnAYzI%3D&reserved=0 > Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217807741&sdata=3YlNePp6l0Gd28HO9T3eZsfLEIAR5sqrGJ8IX7Lbdac%3D&reserved=0 and include the link in your posting. > ***** > > Hi all, > > I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217807741&sdata=36xvdJY5%2Fh8%2B8e7PKdYs8GDBv4Qriv99QEIPBvQtQgc%3D&reserved=0 När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>. När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/ E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy |
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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 Jeremy 1) and 2) yes sorry I took some shortcuts. I should have written 'similar intensity and contrast', not 'quality' which doesn’t mean anything. Of course it is totally possible to find settings on a widefield system that will lead to more bleaching and on a confocal to less bleaching. The learning aim of our workshop is to demonstrate that different fluorophores behave differently and that the fluorophore environment influences this. What we are comparing is images of 4 different samples on a single system. We set our parameters for each system to get bright and nicely contrasted images of our thin samples but of course the images are not comparable between systems. However as an additional outcome of the workshop, we also show, in a completely not quantitative manner, that acquiring bright and contrasted images of the same 4 samples on 4 different imaging modalities is possible as long as the sample is thin and that it leads to very different bleaching behaviour. 3) I totally agree. Although demonstrating bleaching talks a lot to our students about light toxicity for live samples so it is useful to make them aware that they are frying their live cells. I totally agree with what you say: the most important in a microscopy experiment is to get meaningful data and not to preserve the sample. If the imaging itself is not affected by bleaching (e.g end of a z stack) or phototoxicity (live cells), we also recommend to push lasers to diminish the proportion of shot noise and possibly allow for faster imaging and lowering of other sources of noise (e.g. lowering the gain). As you said, never mind if that fixed cells is bleached to death. What is important is that I get my meaningful data. Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeremy Adler Sent: 21 October 2019 12:29 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=tJMIRjSZkUjWkLxI9oYOOwMRQI4c5F8QTqghi1CL%2BqE%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=jZCylVK49cZqfkyA7X1die4iKYp7unRRJeZkmSnU4gE%3D&reserved=0 and include the link in your posting. ***** Hej Sylvie, A couple of thoughts 1) "We set the acquisition parameters to get similar image qualities on all systems" Same resolution, photon counts and image acquisition rates ? difficult. 2) most confocals and multiphoton microscopes are point scanning, so a continuous scan of a large area and a small area will produce an apparent differences in the bleaching rate. 3) bleaching per se is not the real issue, instead we perhaps should consider how many photons are detected for a given amount of photobleaching - how efficient are different microscopes at obtaining photons. A more extreme case is that for a fixed specimen and depending on our question, it doesn’t matter if the sample is finally fully photobleached - since this actually means we have obtained the best possible image - all the available photons have been captured and my slide has 5 000 more cells I can image. 4) Scan speed - is it for a given amount of photobleaching, better to use one slow scan or to average/integrate several faster scans adding up to the same scan time. The multiple fast scans involve generating more pixels, with presumably some additional noise, but since the scan is faster the local excitation power is lower and some systems, live tissue and mountants with antifade agents, should be cope better as their protection will not be overwhelmed by a higher peak power. So faster scanning should be better. I am not arguing that confocals do not produce bleaching, only that our measures of bleaching and the importance of bleaching to our question can be highly variable. Jeremy Adler -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader Sent: den 21 oktober 2019 11:35 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=tJMIRjSZkUjWkLxI9oYOOwMRQI4c5F8QTqghi1CL%2BqE%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=jZCylVK49cZqfkyA7X1die4iKYp7unRRJeZkmSnU4gE%3D&reserved=0 and include the link in your posting. ***** Interesting discussion 😊 During our yearly 1-month course (Thanks Steffen for the clarification! It makes sense!), we have a workshop called The art of bleaching. We prepare non-confluent flat cells on coverslips and mounted on a slide. The cells are stained with a tubulin antibody and we use a secondary with FITC or Alexa Fluor 488. We mount the cells in either PBS (with nail polish) or Prolong Diamond. Then we go with our 4 samples to 4 different imaging modalities (4 student groups in parallel): widefield, spinning disk, single point confocal with galvo scanner, single point confocal with resonant scanner (averaging 4x). We first image continuously (i.e. we record a time lapse with no delay between images) for 3 min, then we immediately start a 2 min timer and when it beeps we acquire 1 more image (at the same exact position). Finally we remove the background everywhere and we compute the following: - intensity at start - intensity after 3 min continuous image or time to reach 50% bleaching - intensity of the final image after 2 min in the dark We set the acquisition parameters to get similar image qualities on all systems. Very fun! The results are: - with a spinning disk or a widefield system, we are not nearly closed to 50% bleaching after 3 min of continuous imaging - with a single point confocal, galvo or resonant, we reach 50% bleaching before the end of the 3 min - the 2 fluorophores behave differently and the mounting medium has a strong influence: FITC in prolong diamond bleaches much slower than AF488 in diamond. It is the opposite for PBS. - the intensity of FITC in PBS is brighter after 2 min in the dark Conclusions: - single point confocals bleach tons. This means that live cells will suffer a lot more than on a widefield/spinning disk - Never scanning continuously while setting the parameters. Take one image, assess, change the settings, take another image... a quick continuous scanning can be used to refocus. Original focusing should be done with bright field. - different fluorophores, even with the same colour, have different photophysics - the mounting medium is important to prevent bleaching. Choose a mounting medium that works for your fluorophore. - when the sample fluorescence goes down, the fluorophores could be bleaching or they could go to the dark state (from which they recover with time) Nothing surprising. All this is expected but demonstrating it during the course makes it go home and it is a lot of fun! One could indefinitely demonstrate all sorts of stuff with this set up, including laser power vs scan speed, line vs frame averaging... Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Steffen Dietzel Sent: 21 October 2019 10:37 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=tJMIRjSZkUjWkLxI9oYOOwMRQI4c5F8QTqghi1CL%2BqE%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208097140&sdata=jZCylVK49cZqfkyA7X1die4iKYp7unRRJeZkmSnU4gE%3D&reserved=0 and include the link in your posting. ***** Hi Timothy, the spinning-disk people say that the critical point about bleaching is maximum peak power, and I tend to believe it. I think what you describe could be true for cw lasers, but maybe not for pulsed lasers, where the fluors have more time to drop back to ground state before the next excitation hits. In any case important would be laser power. But I admit I did not thoroughly test this. And should it really make a difference whether the pixel dwell time is 0.5 or 1 or 2 µs? In both cases there is ample time to go to T-states. Resonant scanning with times <50 ns may be different. Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? I encourage our users to test for bleaching by recording the same image (stack) twice and check for intensity decrease in the second stack. If there is none, time can be save by higher laser power (up to the point of saturation). Fortunately not many of our users use FITC or APC, and modern fluors are pretty stable. Steffen Am 18.10.2019 um 16:16 schrieb Feinstein, Timothy N: > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists > .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Cs > ylvie.le.guyader%40KI.SE%7Ce3dfd0cee8134187f2df08d756024cc9%7Cbff7eef1 > cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072440217797784&sdata=OCCLfa > u9LMQ8l%2B8HFXjh1oALjhho6jIWk%2FtXasnAYzI%3D&reserved=0 > Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208107135&sdata=PQ10GyJ2iVWNkhEZeJiilp0Gj0K1WLlH%2BrbwF9Rg1MA%3D&reserved=0 and include the link in your posting. > ***** > > Hi all, > > I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Biomedical Center (BMC) Head of the Core Facility Bioimaging Großhaderner Straße 9 D-82152 Planegg-Martinsried Germany https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioimaging.bmc.med.uni-muenchen.de&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7Ceab2247906e140852eaa08d756129999%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072510208107135&sdata=LjEvBaZIj1e4IznwiUUMKcOKP1KbU3V1Kgra1C9Alj0%3D&reserved=0 När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>. 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In reply to this post by Jeremy Adler-4
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Steffen, that is the nut of my question. >> Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan?>> I have tested this with gas and solid state lasers and as far as I can tell —strictly from my own experience!— you get more sample loss by slowing the scan than if you raise laser power to get a comparable increase in signal. However I’d gladly submit to any properly done testing out there. In principle if a 100 MHz laser has a 10 ns pulse interval, that’s short enough to doubly excite most common fluors. It seems reasonable then to think that a solid state laser could also endanger the sample more by scanning slowly than by scanning fast with a higher pulse amplitude. Best, T Timothy Feinstein, Ph.D. > On Oct 21, 2019, at 6:37 AM, Jeremy Adler <[hidden email]> wrote: > > Has anyone ever published a paper on this? Fast scan speed and averaging or higher laser power versus slow scan? |
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In reply to this post by Sylvie Le Guyader
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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. ***** Last week I posted a rule to always use a pinhole of 1 Airy unit. There were replies pro and con and why under what circumstances, and I posted that sometimes we do open the pinhole for certain applications. Back in June I collected images of a very bright and stable sample to use to illustrate pinhole performance, but I didn’t go back and look at them. Because of the discussion here, today during lunch I revisited them and was surprised to see how much the resolution varied. Makes me think the rule for routine work should be, "Always set pinhole 0.5 to 1." The images are now online at http://microscopynotes.com/880/pinhole/ Cheers- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 Office: 646-501-0567 Cell: 914-309-3270 [hidden email] -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader Sent: Monday, October 21, 2019 4:34 AM To: [hidden email] Subject: Re: scan speed Our rationale for scan speed, averaging... is: - what is your scientific question: i.e. how will you analyse the images and what is the smallest distance that you need to measure in the sample (distance between objects, not size of objects)? - First choice is widefield, second spinning disk confocal, third single point confocal. And light sheet for large samples whenever we can avoid sectioning. - on a single point confocal, set everything to go as fast as possible (large pixels, fastest scan speed, no averaging). - does the acquired image allow you to answer your scientific question (measure the distance defined above)? - if yes, keep it that way. Set the machine to image automatically lots of sample and go and do something else while it works for you. 😊 - If not, if the pixel size is a problem, check if the chosen objective delivers a resolution that answers the scientific question. Check what the Nyquist pixel size is. Make a decision. Then check the noise by scanning continuously (which we normally never do. We only use snap) and seeing what changes from one image to the next: that is random noise. Random noise can be helped by lowering the gain (increasing lasers if possible), averaging and/or allowing more photons to the detector (more laser if possible, slower scan speed if possible, maybe using a lower magnification with high NA) - If the image is meant to be printed in a paper instead of being for data acquisition, just blast the thing with small pixels, high averaging, high laser... Just get 1 lovely image that way that can be magnified in case you get the journal cover then go back to acquiring 'real' data where all settings are chosen to reliably answer the scientific question. Any comments on our approach are very welcome. 😊 Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Brian Armstrong Sent: 18 October 2019 19:22 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email] Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. 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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 noticed on my Zeiss LSM880 that when I use Airyscan mode the software chooses its own scan speed when you select "optimal" for the pixel dimension. What I found interesting is that when I chose a 63X/1.4 NA lens the pixels were 1380 x 1380 and the scan speed was 5 or 3.04usec/pixel. When I chose a 10x/0.45 NA lens the pixels were 2792 x 2792 and the scan speed was 4 or 3.01usec/pixel. The point being that the Zeiss engineers have made a default scan speed of ~3usec/pixel for Airyscan imaging. This is much slower than the 1.0usec/pixel I typically use. Perhaps Zeiss has tested this speed and determined it provides the best resolution? Perhaps this is a mathematically optimized scan speed? Dr Feinstein reported a scan speed of 700Hz on a Leica system I believe. May I ask, how does this speed equate to u/pixel speeds? Thank you, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael Sent: Monday, October 21, 2019 1:43 PM To: [hidden email] Subject: Re: scan speed ***** 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. ***** Last week I posted a rule to always use a pinhole of 1 Airy unit. There were replies pro and con and why under what circumstances, and I posted that sometimes we do open the pinhole for certain applications. Back in June I collected images of a very bright and stable sample to use to illustrate pinhole performance, but I didn’t go back and look at them. Because of the discussion here, today during lunch I revisited them and was surprised to see how much the resolution varied. Makes me think the rule for routine work should be, "Always set pinhole 0.5 to 1." The images are now online at http://microscopynotes.com/880/pinhole/ Cheers- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 Office: 646-501-0567 Cell: 914-309-3270 [hidden email] -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader Sent: Monday, October 21, 2019 4:34 AM To: [hidden email] Subject: Re: scan speed Our rationale for scan speed, averaging... is: - what is your scientific question: i.e. how will you analyse the images and what is the smallest distance that you need to measure in the sample (distance between objects, not size of objects)? - First choice is widefield, second spinning disk confocal, third single point confocal. And light sheet for large samples whenever we can avoid sectioning. - on a single point confocal, set everything to go as fast as possible (large pixels, fastest scan speed, no averaging). - does the acquired image allow you to answer your scientific question (measure the distance defined above)? - if yes, keep it that way. Set the machine to image automatically lots of sample and go and do something else while it works for you. - If not, if the pixel size is a problem, check if the chosen objective delivers a resolution that answers the scientific question. Check what the Nyquist pixel size is. Make a decision. Then check the noise by scanning continuously (which we normally never do. We only use snap) and seeing what changes from one image to the next: that is random noise. Random noise can be helped by lowering the gain (increasing lasers if possible), averaging and/or allowing more photons to the detector (more laser if possible, slower scan speed if possible, maybe using a lower magnification with high NA) - If the image is meant to be printed in a paper instead of being for data acquisition, just blast the thing with small pixels, high averaging, high laser... Just get 1 lovely image that way that can be magnified in case you get the journal cover then go back to acquiring 'real' data where all settings are chosen to reliably answer the scientific question. Any comments on our approach are very welcome. 😊 Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Brian Armstrong Sent: 18 October 2019 19:22 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email] Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. 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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. ***** Point scanning systems are (hopefully) shot noise limited, so what (hopefully) matters is the number of photons per pixel. If you scan fast at high power, or slow at lower power, or fast at low power but average and in each case get (in expectation) N photons per pixel, you have an SNR of N^0.5 in all instances. If you want more SNR, you must get more photons and it won't matter how you do that. Scanning speed becomes a factor in shot noise limited confocal systems however because detectors have non-zero dark count rate and because they have a maximum detection rate before they saturate. Since dark counts and photon counts both contribute shot noise, if you try to image with a dwell time that isn't much less than the inverse of your dark count rate, the darker pixels in your image will start to be dominated by dark shot noise rather than photon shot noise. In this case you should either image faster or get a detector with fewer dark counts. The maximum detection rate similarly puts an upper bound on how fast you can image for a given SNR by limiting the number of photons any pixel can contain. For example, with a hypothetical detector that has 1 dark count per microsecond on average and a maximum detection rate of 100 photons per microsecond, you would want to image with a dwell time of less than 1 microsecond, and would be limited to a maximum SNR of less than 10. This would be a very bad detector since its dark count rate and maximum detection rate are very close. For real PMTs, usually the dark count rate is extremely low (KHz) while the maximum count rate is somewhere around a few billion, so you can image very slow without being limited by dark counts, but if you try to image with dwell times of much less than a microsecond, your SNR pretty quickly drops down into the 20s or below and you either have to slow down or start averaging. This is why the default dwell time is usually around a few microseconds when using a PMT-based confocal or 2P. -- Michael Giacomelli, Ph.D. Assistant Professor Department of Biomedical Engineering University of Rochester On Mon, Oct 21, 2019 at 5:21 PM Brian Armstrong <[hidden email]<mailto:[hidden email]>> wrote: ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= and include the link in your posting. ***** Hi All, I noticed on my Zeiss LSM880 that when I use Airyscan mode the software chooses its own scan speed when you select "optimal" for the pixel dimension. What I found interesting is that when I chose a 63X/1.4 NA lens the pixels were 1380 x 1380 and the scan speed was 5 or 3.04usec/pixel. When I chose a 10x/0.45 NA lens the pixels were 2792 x 2792 and the scan speed was 4 or 3.01usec/pixel. The point being that the Zeiss engineers have made a default scan speed of ~3usec/pixel for Airyscan imaging. This is much slower than the 1.0usec/pixel I typically use. Perhaps Zeiss has tested this speed and determined it provides the best resolution? Perhaps this is a mathematically optimized scan speed? Dr Feinstein reported a scan speed of 700Hz on a Leica system I believe. May I ask, how does this speed equate to u/pixel speeds? Thank you, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]<mailto:[hidden email]>] On Behalf Of Cammer, Michael Sent: Monday, October 21, 2019 1:43 PM To: [hidden email]<mailto:[hidden email]> Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= and include the link in your posting. ***** Last week I posted a rule to always use a pinhole of 1 Airy unit. There were replies pro and con and why under what circumstances, and I posted that sometimes we do open the pinhole for certain applications. Back in June I collected images of a very bright and stable sample to use to illustrate pinhole performance, but I didn’t go back and look at them. Because of the discussion here, today during lunch I revisited them and was surprised to see how much the resolution varied. Makes me think the rule for routine work should be, "Always set pinhole 0.5 to 1." The images are now online at https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_880_pinhole_&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=azPLr_8NJAfQHfs95awKyR8KVIUP3hnfpczkp-6fSvk&e= Cheers- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 Office: 646-501-0567 Cell: 914-309-3270 [hidden email]<mailto:[hidden email]> -----Original Message----- From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf Of Sylvie Le Guyader Sent: Monday, October 21, 2019 4:34 AM To: [hidden email]<mailto:[hidden email]> Subject: Re: scan speed Our rationale for scan speed, averaging... is: - what is your scientific question: i.e. how will you analyse the images and what is the smallest distance that you need to measure in the sample (distance between objects, not size of objects)? - First choice is widefield, second spinning disk confocal, third single point confocal. And light sheet for large samples whenever we can avoid sectioning. - on a single point confocal, set everything to go as fast as possible (large pixels, fastest scan speed, no averaging). - does the acquired image allow you to answer your scientific question (measure the distance defined above)? - if yes, keep it that way. Set the machine to image automatically lots of sample and go and do something else while it works for you. - If not, if the pixel size is a problem, check if the chosen objective delivers a resolution that answers the scientific question. Check what the Nyquist pixel size is. Make a decision. Then check the noise by scanning continuously (which we normally never do. We only use snap) and seeing what changes from one image to the next: that is random noise. Random noise can be helped by lowering the gain (increasing lasers if possible), averaging and/or allowing more photons to the detector (more laser if possible, slower scan speed if possible, maybe using a lower magnification with high NA) - If the image is meant to be printed in a paper instead of being for data acquisition, just blast the thing with small pixels, high averaging, high laser... Just get 1 lovely image that way that can be magnified in case you get the journal cover then go back to acquiring 'real' data where all settings are chosen to reliably answer the scientific question. Any comments on our approach are very welcome. 😊 Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf Of Brian Armstrong Sent: 18 October 2019 19:22 To: [hidden email]<mailto:[hidden email]> Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]<mailto:[hidden email]>] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email]<mailto:[hidden email]> Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** To join, leave or search the confocal microscopy listserv, go to: https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= Post images on https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. (LCP301) ------------------------------------------------------------ När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_medarbetare_integritetsskyddspolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=p6edQboq1iVC14_uTCKrcxKPrAD96GPiFUhaRXK-fS4&e= >. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_en_staff_data-2Dprotection-2Dpolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=OBXvimZ49OE9APjPqrmr8oErR2Jk1qaeLMk5WToHAFs&e= >. -- Michael Giacomelli, Ph.D. Assistant Professor Department of Biomedical Engineering University of Rochester |
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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 Michael, the concern of one of the early posts in this thread was fluorophore saturation and (superlinear) bleaching. Point scanning confocal is able to saturate the fluorophores in the focus of the laser beam, so 1) the fluorescence intensity is lower than it should be and 2) bleaching may be accelerated (beyond the linear case you've analyzed in detail). With this in mind it makes difference if you scan slowly or quickly (+averaging). And what laser power you use. Since fluorescence itself is a fast process (few nanoseconds), you can't 'outcompete' it by fast scanning (i.e. dwell time of few ns per diffraction spot). And it's not even desirable, you want to collect the fluorescence before moving to the next spot... So it seems to me that fluorophore saturation and the (earlier mentioned) excited state absorption and resulting bleaching can only be reduced by reducing the peak intensity (e.g. by parallelization = spinning disk). But there are other transient states of the molecules with microsecond - millisecond lifetimes that can lead to increased bleaching (when hit by yet another photon), and this is where faster scanning (+averaging) may help. I say *may* as I haven't performed in-depth experimental testing of these effects. And, they will be heavily sample-dependent... Best, zdenek On Mon, Oct 21, 2019 at 11:50 PM Michael Giacomelli < [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. > ***** > > Point scanning systems are (hopefully) shot noise limited, so what > (hopefully) matters is the number of photons per pixel. If you scan fast > at high power, or slow at lower power, or fast at low power but average and > in each case get (in expectation) N photons per pixel, you have an SNR of > N^0.5 in all instances. If you want more SNR, you must get more photons > and it won't matter how you do that. > > Scanning speed becomes a factor in shot noise limited confocal systems > however because detectors have non-zero dark count rate and because they > have a maximum detection rate before they saturate. Since dark counts and > photon counts both contribute shot noise, if you try to image with a dwell > time that isn't much less than the inverse of your dark count rate, the > darker pixels in your image will start to be dominated by dark shot noise > rather than photon shot noise. In this case you should either image faster > or get a detector with fewer dark counts. The maximum detection rate > similarly puts an upper bound on how fast you can image for a given SNR by > limiting the number of photons any pixel can contain. For example, with a > hypothetical detector that has 1 dark count per microsecond on average and > a maximum detection rate of 100 photons per microsecond, you would want to > image with a dwell time of less than 1 microsecond, and would be limited to > a maximum SNR of less than 10. This would be a very bad detector since its > dark count rate and maximum detection rate are very close. > > For real PMTs, usually the dark count rate is extremely low (KHz) while > the maximum count rate is somewhere around a few billion, so you can image > very slow without being limited by dark counts, but if you try to image > with dwell times of much less than a microsecond, your SNR pretty quickly > drops down into the 20s or below and you either have to slow down or start > averaging. This is why the default dwell time is usually around a few > microseconds when using a PMT-based confocal or 2P. > > -- > Michael Giacomelli, Ph.D. > Assistant Professor > Department of Biomedical Engineering > University of Rochester > > On Mon, Oct 21, 2019 at 5:21 PM Brian Armstrong <[hidden email] > <mailto:[hidden email]>> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= > Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= > and include the link in your posting. > ***** > > Hi All, I noticed on my Zeiss LSM880 that when I use Airyscan mode the > software chooses its own scan speed when you select "optimal" for the pixel > dimension. What I found interesting is that when I chose a 63X/1.4 NA lens > the pixels were 1380 x 1380 and the scan speed was 5 or 3.04usec/pixel. > When I chose a 10x/0.45 NA lens the pixels were 2792 x 2792 and the scan > speed was 4 or 3.01usec/pixel. The point being that the Zeiss engineers > have made a default scan speed of ~3usec/pixel for Airyscan imaging. This > is much slower than the 1.0usec/pixel I typically use. Perhaps Zeiss has > tested this speed and determined it provides the best resolution? Perhaps > this is a mathematically optimized scan speed? > > Dr Feinstein reported a scan speed of 700Hz on a Leica system I believe. > May I ask, how does this speed equate to u/pixel speeds? > > Thank you, > > Brian Armstrong PhD > Associate Research Professor > Developmental and Stem Cell Biology > Diabetes and Metabolic Diseases > Director, Light Microscopy Core > Beckman Research Institute, City of Hope > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email] > <mailto:[hidden email]>] On Behalf Of Cammer, Michael > Sent: Monday, October 21, 2019 1:43 PM > To: [hidden email]<mailto: > [hidden email]> > Subject: Re: scan speed > > ***** > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= > Post images on > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= > and include the link in your posting. > ***** > > Last week I posted a rule to always use a pinhole of 1 Airy unit. There > were replies pro and con and why under what circumstances, and I posted > that sometimes we do open the pinhole for certain applications. > > Back in June I collected images of a very bright and stable sample to use > to illustrate pinhole performance, but I didn’t go back and look at them. > Because of the discussion here, today during lunch I revisited them and was > surprised to see how much the resolution varied. Makes me think the rule > for routine work should be, "Always set pinhole 0.5 to 1." The images are > now online at > https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_880_pinhole_&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=azPLr_8NJAfQHfs95awKyR8KVIUP3hnfpczkp-6fSvk&e= > > Cheers- > > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU > Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 > Office: 646-501-0567 Cell: 914-309-3270 [hidden email] > <mailto:[hidden email]> > > > > > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]<mailto: > [hidden email]>> On Behalf Of Sylvie Le Guyader > Sent: Monday, October 21, 2019 4:34 AM > To: [hidden email]<mailto: > [hidden email]> > Subject: Re: scan speed > > > Our rationale for scan speed, averaging... is: > - what is your scientific question: i.e. how will you analyse the images > and what is the smallest distance that you need to measure in the sample > (distance between objects, not size of objects)? > - First choice is widefield, second spinning disk confocal, third single > point confocal. And light sheet for large samples whenever we can avoid > sectioning. > - on a single point confocal, set everything to go as fast as possible > (large pixels, fastest scan speed, no averaging). > - does the acquired image allow you to answer your scientific question > (measure the distance defined above)? > - if yes, keep it that way. Set the machine to image automatically lots of > sample and go and do something else while it works for you. > - If not, if the pixel size is a problem, check if the chosen objective > delivers a resolution that answers the scientific question. Check what the > Nyquist pixel size is. Make a decision. Then check the noise by scanning > continuously (which we normally never do. We only use snap) and seeing what > changes from one image to the next: that is random noise. Random noise can > be helped by lowering the gain (increasing lasers if possible), averaging > and/or allowing more photons to the detector (more laser if possible, > slower scan speed if possible, maybe using a lower magnification with high > NA) > - If the image is meant to be printed in a paper instead of being for data > acquisition, just blast the thing with small pixels, high averaging, high > laser... Just get 1 lovely image that way that can be magnified in case you > get the journal cover then go back to acquiring 'real' data where all > settings are chosen to reliably answer the scientific question. > > Any comments on our approach are very welcome. 😊 > > Med vänlig hälsning / Best regards > > Sylvie > > @@@@@@@@@@@@@@@@@@@@@@@@ > Sylvie Le Guyader, PhD > Live Cell Imaging Facility Manager > Karolinska Institutet- Bionut Dpt > Blickagången 16, > Room 7362 (lab)/7840 (office) > 14157 Huddinge, Sweden > mobile: +46 (0) 73 733 5008 > LCI website > Follow our microscopy blog! > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]<mailto: > [hidden email]>> On Behalf Of Brian Armstrong > Sent: 18 October 2019 19:22 > To: [hidden email]<mailto: > [hidden email]> > Subject: Re: scan speed > > ***** > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= > Post images on > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= > and include the link in your posting. > ***** > > Hi, well I have always thought that the longer the pixel dwell time the > more accurate the representation the image is of the sample. However, there > must be an optimal scan speed that relates to Nyquist sampling (Nyquist > indeed first theorized about temporal sampling). Perhaps modern electronics > perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that > sampling speed just isn't a factor in modern Confocal imaging as far as the > electronics are concerned. That being said there must be a "sweet spot" for > temporal sampling although it isn't really discussed much. I default to > scan speed of 1.02usec/pixel. I would be happy to hear how and why I am > wrong. > > As far as the number of Prinicipal Investigators at my institution that > would be willing to pay for training of 16 hours on a Confocal (mind you, > our time is free, they simply pay for the instrument time) is approximately > zero. Moreover, in my opinion there isn't a sound reason why each user > needs to have the expertise of a microscope guru. That would negate one key > reason why the core model is valid (the other being instrument cost). > Furthermore, teaching someone the intricacies of z-stack acquisition for 8 > hours who isn't performing z-stacks seems unnecessary (for example). > > Cheers, > > Brian Armstrong PhD > Associate Research Professor > Developmental and Stem Cell Biology > Diabetes and Metabolic Diseases > Director, Light Microscopy Core > Beckman Research Institute, City of Hope > > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email] > <mailto:[hidden email]>] On Behalf Of Feinstein, > Timothy N > Sent: Friday, October 18, 2019 7:17 AM > To: [hidden email]<mailto: > [hidden email]> > Subject: scan speed > > [Attention: This email came from an external source. Do not open > attachments or click on links from unknown senders or unexpected emails.] > > > > > > ***** > To join, leave or search the confocal microscopy listserv, go to: > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= > Post images on > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= > and include the link in your posting. > ***** > > Hi all, > > I can't understate how helpful this discussion about training and best > practices has been. My sincere thanks go to everyone who's participated. > > I’d like to put a finer point on a question about scanning speed. Like > some others I encourage users to just set the fastest speed and leave it > there. Leica SP8s constrain the field of view with faster speed, so I > suggest SP8 users choose 700 Hz as a good balance between speed and field > of view. My reasoning comes strictly from my own testing - I found that > you pay a lot more in bleaching when you try to get a brighter signal by > slowing the scan than if you just turn up the laser power. Since the > slowest speeds drastically increase bleaching, I use them for a bleaching / > photoactivation ROI and that's about it. > > As I understand it, the strong relationship between scan speed and > bleaching has to do with longer dwell time increasing the chance of > exciting fluorophores to the triplet state. Hence why resonant scan plus > line averaging is gentler on a live sample than a galvo scan of comparable > signal-to-noise. Is that right? Thanks! > > > T > > Timothy Feinstein, Ph.D. > Research Scientist > Department of Developmental Biology > University of Pittsburgh > > > > ------------------------------------------------------------ > -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. > (LCP301) > ------------------------------------------------------------ > > > När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI > kommer att behandla dina personuppgifter. Här finns information om hur KI > behandlar personuppgifter< > https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_medarbetare_integritetsskyddspolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=p6edQboq1iVC14_uTCKrcxKPrAD96GPiFUhaRXK-fS4&e= > >. > > > Sending email to Karolinska Institutet (KI) will result in KI processing > your personal data. You can read more about KI’s processing of personal > data here< > https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_en_staff_data-2Dprotection-2Dpolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=OBXvimZ49OE9APjPqrmr8oErR2Jk1qaeLMk5WToHAFs&e= > >. > > > -- > Michael Giacomelli, Ph.D. > Assistant Professor > Department of Biomedical Engineering > University of Rochester > -- -- Zdenek Svindrych, Ph.D. Research Associate - Imaging Specialist Department of Biochemistry and Cell Biology Geisel School of Medicine at Dartmouth |
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In reply to this post by Cammer, Michael
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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 Michael Thanks for sharing! 😊 I like that spirit! Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Hälsovägen 7C, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Sign in to follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Cammer, Michael Sent: 21 October 2019 22:43 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571886412&sdata=YcD75m34D%2F9PYWUHOERib2N4%2FK2gDivu4rQSlgdrI04%3D&reserved=0 Post images on https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=mluV%2FPcHNjiTuTpwepGbJYzALAhlhX4IQ%2BXmWPfSl9A%3D&reserved=0 and include the link in your posting. ***** Last week I posted a rule to always use a pinhole of 1 Airy unit. There were replies pro and con and why under what circumstances, and I posted that sometimes we do open the pinhole for certain applications. Back in June I collected images of a very bright and stable sample to use to illustrate pinhole performance, but I didn’t go back and look at them. Because of the discussion here, today during lunch I revisited them and was surprised to see how much the resolution varied. Makes me think the rule for routine work should be, "Always set pinhole 0.5 to 1." The images are now online at https://eur01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicroscopynotes.com%2F880%2Fpinhole%2F&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=w0GVeUhYtPMW6cRneDNsVaVS7Qe%2BFn0%2BNfiL%2BT7ZkCs%3D&reserved=0 Cheers- Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016 Office: 646-501-0567 Cell: 914-309-3270 [hidden email] -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Sylvie Le Guyader Sent: Monday, October 21, 2019 4:34 AM To: [hidden email] Subject: Re: scan speed Our rationale for scan speed, averaging... is: - what is your scientific question: i.e. how will you analyse the images and what is the smallest distance that you need to measure in the sample (distance between objects, not size of objects)? - First choice is widefield, second spinning disk confocal, third single point confocal. And light sheet for large samples whenever we can avoid sectioning. - on a single point confocal, set everything to go as fast as possible (large pixels, fastest scan speed, no averaging). - does the acquired image allow you to answer your scientific question (measure the distance defined above)? - if yes, keep it that way. Set the machine to image automatically lots of sample and go and do something else while it works for you. 😊 - If not, if the pixel size is a problem, check if the chosen objective delivers a resolution that answers the scientific question. Check what the Nyquist pixel size is. Make a decision. Then check the noise by scanning continuously (which we normally never do. We only use snap) and seeing what changes from one image to the next: that is random noise. Random noise can be helped by lowering the gain (increasing lasers if possible), averaging and/or allowing more photons to the detector (more laser if possible, slower scan speed if possible, maybe using a lower magnification with high NA) - If the image is meant to be printed in a paper instead of being for data acquisition, just blast the thing with small pixels, high averaging, high laser... Just get 1 lovely image that way that can be magnified in case you get the journal cover then go back to acquiring 'real' data where all settings are chosen to reliably answer the scientific question. Any comments on our approach are very welcome. 😊 Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader, PhD Live Cell Imaging Facility Manager Karolinska Institutet- Bionut Dpt Blickagången 16, Room 7362 (lab)/7840 (office) 14157 Huddinge, Sweden mobile: +46 (0) 73 733 5008 LCI website Follow our microscopy blog! -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Brian Armstrong Sent: 18 October 2019 19:22 To: [hidden email] Subject: Re: scan speed ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3Dj5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs%26r%3DE0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU%26m%3DipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw%26s%3Do22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ%26e&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=KY4Nlx%2FVJ0sqLBmxWlNAKBUvwaJPnj75FgQ3oNWmCwk%3D&reserved=0= Post images on https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3Dj5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs%26r%3DE0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU%26m%3DipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw%26s%3DCQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8%26e&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=wRiskgZtknnOEmlBaDCJPaq87yh7HCXENUdIxTI4cfc%3D&reserved=0= and include the link in your posting. ***** Hi, well I have always thought that the longer the pixel dwell time the more accurate the representation the image is of the sample. However, there must be an optimal scan speed that relates to Nyquist sampling (Nyquist indeed first theorized about temporal sampling). Perhaps modern electronics perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly that sampling speed just isn't a factor in modern Confocal imaging as far as the electronics are concerned. That being said there must be a "sweet spot" for temporal sampling although it isn't really discussed much. I default to scan speed of 1.02usec/pixel. I would be happy to hear how and why I am wrong. As far as the number of Prinicipal Investigators at my institution that would be willing to pay for training of 16 hours on a Confocal (mind you, our time is free, they simply pay for the instrument time) is approximately zero. Moreover, in my opinion there isn't a sound reason why each user needs to have the expertise of a microscope guru. That would negate one key reason why the core model is valid (the other being instrument cost). Furthermore, teaching someone the intricacies of z-stack acquisition for 8 hours who isn't performing z-stacks seems unnecessary (for example). Cheers, Brian Armstrong PhD Associate Research Professor Developmental and Stem Cell Biology Diabetes and Metabolic Diseases Director, Light Microscopy Core Beckman Research Institute, City of Hope -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 18, 2019 7:17 AM To: [hidden email] Subject: scan speed [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.] ***** To join, leave or search the confocal microscopy listserv, go to: https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3Dj5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs%26r%3DE0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU%26m%3DipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw%26s%3Do22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ%26e&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=KY4Nlx%2FVJ0sqLBmxWlNAKBUvwaJPnj75FgQ3oNWmCwk%3D&reserved=0= Post images on https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0%26d%3DDwIGaQ%26c%3Dj5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs%26r%3DE0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU%26m%3DipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw%26s%3DCQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8%26e&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571896402&sdata=wRiskgZtknnOEmlBaDCJPaq87yh7HCXENUdIxTI4cfc%3D&reserved=0= and include the link in your posting. ***** Hi all, I can't understate how helpful this discussion about training and best practices has been. My sincere thanks go to everyone who's participated. I’d like to put a finer point on a question about scanning speed. Like some others I encourage users to just set the fastest speed and leave it there. Leica SP8s constrain the field of view with faster speed, so I suggest SP8 users choose 700 Hz as a good balance between speed and field of view. My reasoning comes strictly from my own testing - I found that you pay a lot more in bleaching when you try to get a brighter signal by slowing the scan than if you just turn up the laser power. Since the slowest speeds drastically increase bleaching, I use them for a bleaching / photoactivation ROI and that's about it. As I understand it, the strong relationship between scan speed and bleaching has to do with longer dwell time increasing the chance of exciting fluorophores to the triplet state. Hence why resonant scan plus line averaging is gentler on a live sample than a galvo scan of comparable signal-to-noise. Is that right? Thanks! T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh ------------------------------------------------------------ -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. (LCP301) ------------------------------------------------------------ När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Furldefense.proofpoint.com%2Fv2%2Furl%3Fu%3Dhttps-3A__ki.se_medarbetare_integritetsskyddspolicy%26d%3DDwIGaQ%26c%3Dj5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs%26r%3DE0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU%26m%3DipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw%26s%3Dp6edQboq1iVC14_uTCKrcxKPrAD96GPiFUhaRXK-fS4%26e&data=02%7C01%7Csylvie.le.guyader%40KI.SE%7C2ff122b53abe4dd7bde108d756676f3c%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C0%7C637072874571906395&sdata=FuYyisZutXCUIyX1etnzwGFAVT3tM13HmrqlsElGIjs%3D&reserved=0= >. Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. 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In reply to this post by Zdenek Svindrych-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. ***** My understanding has always been that fast scanning would help, but we can't go fast enough with current commercial equipment. This was shown by Stefan Hell's lab when they used an EOD to obtain an pixel dwell time of ~6 ns. This means a single excitation event/fluorophore/scan. Here are some relevant quotes from the paper: "In confocal microscopes with approximately microsecond pixel dwell times, fluorophores typically face 10–1,000 excitation events until the illumination spot is moved, usually after a certain number of photons are collected on average. Thus, the excitation, detection and bleaching events appear continuous despite the stochastic nature of these processes. Only if the dwell time of the moving illumination spot on a fluorophore is shorter than the average pause between two excitation events will the molecule not be subjected to multiple events. In this case, the molecule will emit at most one photon per illumination cycle, preserving the stochastic nature of the emission from the interrogated pixel. In the simple but common situation of excitation with relatively bright pulses that are shorter than the fluorescent state lifetime, the light exposure of a molecule typically must be shorter than the interval between two pulses." "Therefore, our fast scanning approach reduces bleaching and blinking. In fact, we compared the fluorescence yield of new ultrafast scanning with that of conventional (slow) scanning by imaging equally sized and dense areas for equal durations. For many fluorophores and laser configurations, we observed that the total signal increased by 1.5- to 4.5-fold when ultrafast scanning was used (Supplementary Fig. 1)." From: Schneider et al., Nature Methods 2015: https://nanobiophotonics.mpibpc.mpg.de/media/papers/Nature_Meth._12_9_827-830.pdf -Doug On Tue, Oct 22, 2019 at 12:34 AM Zdenek Svindrych <[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 Michael, > > the concern of one of the early posts in this thread was fluorophore > saturation and (superlinear) bleaching. > Point scanning confocal is able to saturate the fluorophores in the focus > of the laser beam, so 1) the fluorescence intensity is lower than it should > be and 2) bleaching may be accelerated (beyond the linear case you've > analyzed in detail). > With this in mind it makes difference if you scan slowly or quickly > (+averaging). And what laser power you use. > Since fluorescence itself is a fast process (few nanoseconds), you can't > 'outcompete' it by fast scanning (i.e. dwell time of few ns per diffraction > spot). And it's not even desirable, you want to collect the fluorescence > before moving to the next spot... So it seems to me that fluorophore > saturation and the (earlier mentioned) excited state absorption and > resulting bleaching can only be reduced by reducing the peak intensity > (e.g. by parallelization = spinning disk). > But there are other transient states of the molecules with microsecond - > millisecond lifetimes that can lead to increased bleaching (when hit by yet > another photon), and this is where faster scanning (+averaging) may help. > > I say *may* as I haven't performed in-depth experimental testing of these > effects. And, they will be heavily sample-dependent... > > Best, zdenek > > On Mon, Oct 21, 2019 at 11:50 PM Michael Giacomelli < > [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. > > ***** > > > > Point scanning systems are (hopefully) shot noise limited, so what > > (hopefully) matters is the number of photons per pixel. If you scan fast > > at high power, or slow at lower power, or fast at low power but average > and > > in each case get (in expectation) N photons per pixel, you have an SNR of > > N^0.5 in all instances. If you want more SNR, you must get more photons > > and it won't matter how you do that. > > > > Scanning speed becomes a factor in shot noise limited confocal systems > > however because detectors have non-zero dark count rate and because they > > have a maximum detection rate before they saturate. Since dark counts > and > > photon counts both contribute shot noise, if you try to image with a > dwell > > time that isn't much less than the inverse of your dark count rate, the > > darker pixels in your image will start to be dominated by dark shot noise > > rather than photon shot noise. In this case you should either image > faster > > or get a detector with fewer dark counts. The maximum detection rate > > similarly puts an upper bound on how fast you can image for a given SNR > by > > limiting the number of photons any pixel can contain. For example, with > a > > hypothetical detector that has 1 dark count per microsecond on average > and > > a maximum detection rate of 100 photons per microsecond, you would want > to > > image with a dwell time of less than 1 microsecond, and would be limited > to > > a maximum SNR of less than 10. This would be a very bad detector since > its > > dark count rate and maximum detection rate are very close. > > > > For real PMTs, usually the dark count rate is extremely low (KHz) while > > the maximum count rate is somewhere around a few billion, so you can > image > > very slow without being limited by dark counts, but if you try to image > > with dwell times of much less than a microsecond, your SNR pretty quickly > > drops down into the 20s or below and you either have to slow down or > start > > averaging. This is why the default dwell time is usually around a few > > microseconds when using a PMT-based confocal or 2P. > > > > -- > > Michael Giacomelli, Ph.D. > > Assistant Professor > > Department of Biomedical Engineering > > University of Rochester > > > > On Mon, Oct 21, 2019 at 5:21 PM Brian Armstrong <[hidden email] > > <mailto:[hidden email]>> wrote: > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > > > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= > > Post images on > > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= > > and include the link in your posting. > > ***** > > > > Hi All, I noticed on my Zeiss LSM880 that when I use Airyscan mode the > > software chooses its own scan speed when you select "optimal" for the > pixel > > dimension. What I found interesting is that when I chose a 63X/1.4 NA > lens > > the pixels were 1380 x 1380 and the scan speed was 5 or 3.04usec/pixel. > > When I chose a 10x/0.45 NA lens the pixels were 2792 x 2792 and the scan > > speed was 4 or 3.01usec/pixel. The point being that the Zeiss engineers > > have made a default scan speed of ~3usec/pixel for Airyscan imaging. This > > is much slower than the 1.0usec/pixel I typically use. Perhaps Zeiss has > > tested this speed and determined it provides the best resolution? Perhaps > > this is a mathematically optimized scan speed? > > > > Dr Feinstein reported a scan speed of 700Hz on a Leica system I believe. > > May I ask, how does this speed equate to u/pixel speeds? > > > > Thank you, > > > > Brian Armstrong PhD > > Associate Research Professor > > Developmental and Stem Cell Biology > > Diabetes and Metabolic Diseases > > Director, Light Microscopy Core > > Beckman Research Institute, City of Hope > > > > > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email] > > <mailto:[hidden email]>] On Behalf Of Cammer, Michael > > Sent: Monday, October 21, 2019 1:43 PM > > To: [hidden email]<mailto: > > [hidden email]> > > Subject: Re: scan speed > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > > > > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=QKO48wggw8Wq37rRsWH7s4FKX2yrcU1JCY04hjUNtN0&e= > > Post images on > > > https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=0S7OdYEhUvIb8br5EkkD9PW2O82sqI02CEf-0YBngEQ&e= > > and include the link in your posting. > > ***** > > > > Last week I posted a rule to always use a pinhole of 1 Airy unit. There > > were replies pro and con and why under what circumstances, and I posted > > that sometimes we do open the pinhole for certain applications. > > > > Back in June I collected images of a very bright and stable sample to use > > to illustrate pinhole performance, but I didn’t go back and look at them. > > Because of the discussion here, today during lunch I revisited them and > was > > surprised to see how much the resolution varied. Makes me think the rule > > for routine work should be, "Always set pinhole 0.5 to 1." The images > are > > now online at > > > https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopynotes.com_880_pinhole_&d=DwIGaQ&c=kbmfwr1Yojg42sGEpaQh5ofMHBeTl9EI2eaqQZhHbOU&r=0LyF_z8oU1XGGyisIeOIXyIGIM5IYb3NcLjxHjUca5Y&m=vavoUD45wXv8Vhg_0EjmxRxd1lA-4GA-tIOimfwl0Ng&s=azPLr_8NJAfQHfs95awKyR8KVIUP3hnfpczkp-6fSvk&e= > > > > Cheers- > > > > Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU > > Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY > 10016 > > Office: 646-501-0567 Cell: 914-309-3270 [hidden email] > > <mailto:[hidden email]> > > > > > > > > > > > > -----Original Message----- > > From: Confocal Microscopy List <[hidden email]<mailto: > > [hidden email]>> On Behalf Of Sylvie Le Guyader > > Sent: Monday, October 21, 2019 4:34 AM > > To: [hidden email]<mailto: > > [hidden email]> > > Subject: Re: scan speed > > > > > > Our rationale for scan speed, averaging... is: > > - what is your scientific question: i.e. how will you analyse the images > > and what is the smallest distance that you need to measure in the sample > > (distance between objects, not size of objects)? > > - First choice is widefield, second spinning disk confocal, third single > > point confocal. And light sheet for large samples whenever we can avoid > > sectioning. > > - on a single point confocal, set everything to go as fast as possible > > (large pixels, fastest scan speed, no averaging). > > - does the acquired image allow you to answer your scientific question > > (measure the distance defined above)? > > - if yes, keep it that way. Set the machine to image automatically lots > of > > sample and go and do something else while it works for you. > > - If not, if the pixel size is a problem, check if the chosen objective > > delivers a resolution that answers the scientific question. Check what > the > > Nyquist pixel size is. Make a decision. Then check the noise by scanning > > continuously (which we normally never do. We only use snap) and seeing > what > > changes from one image to the next: that is random noise. Random noise > can > > be helped by lowering the gain (increasing lasers if possible), averaging > > and/or allowing more photons to the detector (more laser if possible, > > slower scan speed if possible, maybe using a lower magnification with > high > > NA) > > - If the image is meant to be printed in a paper instead of being for > data > > acquisition, just blast the thing with small pixels, high averaging, high > > laser... Just get 1 lovely image that way that can be magnified in case > you > > get the journal cover then go back to acquiring 'real' data where all > > settings are chosen to reliably answer the scientific question. > > > > Any comments on our approach are very welcome. 😊 > > > > Med vänlig hälsning / Best regards > > > > Sylvie > > > > @@@@@@@@@@@@@@@@@@@@@@@@ > > Sylvie Le Guyader, PhD > > Live Cell Imaging Facility Manager > > Karolinska Institutet- Bionut Dpt > > Blickagången 16, > > Room 7362 (lab)/7840 (office) > > 14157 Huddinge, Sweden > > mobile: +46 (0) 73 733 5008 > > LCI website > > Follow our microscopy blog! > > > > -----Original Message----- > > From: Confocal Microscopy List <[hidden email]<mailto: > > [hidden email]>> On Behalf Of Brian Armstrong > > Sent: 18 October 2019 19:22 > > To: [hidden email]<mailto: > > [hidden email]> > > Subject: Re: scan speed > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= > > Post images on > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= > > and include the link in your posting. > > ***** > > > > Hi, well I have always thought that the longer the pixel dwell time the > > more accurate the representation the image is of the sample. However, > there > > must be an optimal scan speed that relates to Nyquist sampling (Nyquist > > indeed first theorized about temporal sampling). Perhaps modern > electronics > > perform analog-to-digital (ADC, and digital-to-analog DAC) so quickly > that > > sampling speed just isn't a factor in modern Confocal imaging as far as > the > > electronics are concerned. That being said there must be a "sweet spot" > for > > temporal sampling although it isn't really discussed much. I default to > > scan speed of 1.02usec/pixel. I would be happy to hear how and why I am > > wrong. > > > > As far as the number of Prinicipal Investigators at my institution that > > would be willing to pay for training of 16 hours on a Confocal (mind you, > > our time is free, they simply pay for the instrument time) is > approximately > > zero. Moreover, in my opinion there isn't a sound reason why each user > > needs to have the expertise of a microscope guru. That would negate one > key > > reason why the core model is valid (the other being instrument cost). > > Furthermore, teaching someone the intricacies of z-stack acquisition for > 8 > > hours who isn't performing z-stacks seems unnecessary (for example). > > > > Cheers, > > > > Brian Armstrong PhD > > Associate Research Professor > > Developmental and Stem Cell Biology > > Diabetes and Metabolic Diseases > > Director, Light Microscopy Core > > Beckman Research Institute, City of Hope > > > > > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email] > > <mailto:[hidden email]>] On Behalf Of Feinstein, > > Timothy N > > Sent: Friday, October 18, 2019 7:17 AM > > To: [hidden email]<mailto: > > [hidden email]> > > Subject: scan speed > > > > [Attention: This email came from an external source. Do not open > > attachments or click on links from unknown senders or unexpected emails.] > > > > > > > > > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Flists.umn.edu-252Fcgi-2Dbin-252Fwa-253FA0-253Dconfocalmicroscopy-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DqwfzHkwdA8phXBub371LRXPjruj24QWtbbmZGTZ8RfM-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=o22pCA-8XENorZTvSBI3BcgBJ0H7DXele_sYn7FBMLQ&e= > > Post images on > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__eur01.safelinks.protection.outlook.com_-3Furl-3Dhttp-253A-252F-252Fwww.imgur.com-26amp-3Bdata-3D02-257C01-257Csylvie.le.guyader-2540KI.SE-257C0481d41463204c15201f08d753efc2cc-257Cbff7eef1cf4b4f32be3da1dda043c05d-257C0-257C0-257C637070161556740296-26amp-3Bsdata-3DKgbaDOG2MLiJQIA-252BL8uH4LGV79BhrfQk5e562tfkG3g-253D-26amp-3Breserved-3D0&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=CQzOxd2t50w8ikNBygnRQTeT65UYEiCnAGtP08Tq3v8&e= > > and include the link in your posting. > > ***** > > > > Hi all, > > > > I can't understate how helpful this discussion about training and best > > practices has been. My sincere thanks go to everyone who's participated. > > > > I’d like to put a finer point on a question about scanning speed. Like > > some others I encourage users to just set the fastest speed and leave it > > there. Leica SP8s constrain the field of view with faster speed, so I > > suggest SP8 users choose 700 Hz as a good balance between speed and field > > of view. My reasoning comes strictly from my own testing - I found that > > you pay a lot more in bleaching when you try to get a brighter signal by > > slowing the scan than if you just turn up the laser power. Since the > > slowest speeds drastically increase bleaching, I use them for a > bleaching / > > photoactivation ROI and that's about it. > > > > As I understand it, the strong relationship between scan speed and > > bleaching has to do with longer dwell time increasing the chance of > > exciting fluorophores to the triplet state. Hence why resonant scan plus > > line averaging is gentler on a live sample than a galvo scan of > comparable > > signal-to-noise. Is that right? Thanks! > > > > > > T > > > > Timothy Feinstein, Ph.D. > > Research Scientist > > Department of Developmental Biology > > University of Pittsburgh > > > > > > > > ------------------------------------------------------------ > > -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. > > (LCP301) > > ------------------------------------------------------------ > > > > > > När du skickar e-post till Karolinska Institutet (KI) innebär detta att > KI > > kommer att behandla dina personuppgifter. Här finns information om hur KI > > behandlar personuppgifter< > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_medarbetare_integritetsskyddspolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=p6edQboq1iVC14_uTCKrcxKPrAD96GPiFUhaRXK-fS4&e= > > >. > > > > > > Sending email to Karolinska Institutet (KI) will result in KI processing > > your personal data. You can read more about KI’s processing of personal > > data here< > > > https://urldefense.proofpoint.com/v2/url?u=https-3A__ki.se_en_staff_data-2Dprotection-2Dpolicy&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedeElZfc04rx3ExJHeIIZuCs&r=E0xNnPAQpUbDiPlC50tp7rW2nBkvV7fujQf0RknE5bU&m=ipYWOfYM7wxroBtcAuzYKT_TyA2rywV77D9bf2EQpXw&s=OBXvimZ49OE9APjPqrmr8oErR2Jk1qaeLMk5WToHAFs&e= > > >. > > > > > > -- > > Michael Giacomelli, Ph.D. > > Assistant Professor > > Department of Biomedical Engineering > > University of Rochester > > > > > -- > -- > Zdenek Svindrych, Ph.D. > Research Associate - Imaging Specialist > Department of Biochemistry and Cell Biology > Geisel School of Medicine at Dartmouth > |
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