Craig,
This makes so much sense of ‘non-descanned’ and ‘descanned’ detectors. Thank you.
Lu
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Craig Brideau
Sent: Wednesday, October 26, 2016 6:47 PM
To: [hidden email]
Subject: Re: Nyquist theorem and DAQ sampling rate, pixel rate, laser rep. rate, and detector/amp bandwidth
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Multi-photon usually uses non-descanned detection, which has the primary dichroic between the scan mirrors and the objective. The fluorescent light from the sample is therefore still moving when it reaches the
detector. As a result it is important that the detector be large so it captures all the light that is moving around. Confocal uses descanned detectors, where the primary dichroic is between the laser and the scan mirrors. In this configuration the fluorescent
light from the sample passes back through the scan mirrors before being collected. This makes the light stationary, which is vital for focusing through a confocal pinhole.
Craig
On Wed, Oct 26, 2016 at 3:16 PM, Yan, Lu <[hidden email]> wrote:
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Andreas,
Thanks much for your answering. Now I get that detector area matters, but what did you mean by non-descanned detectors? Are they not all descanned in multiphoton microscopy like confocal does?
Thanks,
Lu
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Andreas Bruckbauer
Sent: Wednesday, October 26, 2016 4:51 PM
To: [hidden email]
Subject: Re: Nyquist theorem and DAQ sampling rate, pixel rate, laser rep. rate, and detector/amp bandwidth
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Lu,
Your question:
Why single photon counting modules (APD or PMT based) is not as popular as analog PMTs in multiphoton fluorescence microscopy?
The answer is the size (area) of the detector, you want large area detectors to collect all the scattered light, at least when you use two-photon excitation to image deep into tissue with non-descanned detectors.
Best wishes
Andreas
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Sent: 26/10/2016 20:57
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Subject: Nyquist theorem and DAQ sampling rate, pixel rate, laser rep. rate, and detector/amp bandwidth***** 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 listers,
I was reading a multiphoton fluorescence imaging paper earlier today, and found in the method section that the authors used 15 us pixel clock (I assume this is the dwell time, thus the pixel rate is 1/15us=~667 kHz?), a 1 MHz pulsed laser, AO (to drive scanning) sampling rate ~1MHz, but a 20 kHz PMT amp. I got a little confused, so my questions are:
1) Is it generally true that the AO sampling rate has to be at least twice of signal frequency (National Instruments suggested >10X of signal rate), which in this case is limited by the amp rate, i.e. 20kHz? In this paper particularly, they are massively oversampling which is OK I guess.
2) The pixel rate cannot exceed the half of the signal rate (amp bandwidth) to catch all spatial varying signal from the sample. What is considered as appropriate pixel rate given the amp bandwidth? Here, the amp bandwidth is only 20 kHz, but the pixel rate is calculated to be 667 kHz.. Can I assume either they have a typo somewhere, or they compromised the measurement by effectively applying a low pass filter to the images, or I am just being completely idiotic.
3) In general , why single photon counting modules (APD or PMT based) is not as popular as analog PMTs in multiphoton fluorescence microscopy? Do they not have less constrains for example bandwidth than analog ones?
I look forward to hearing from you guys. This really bugged me quite a few hours.
Thanks in advance,
Lu
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