Posted by
Michael Giacomelli on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Nyquist-theorem-and-DAQ-sampling-rate-pixel-rate-laser-rep-rate-and-detector-amp-bandwidth-tp7585926p7585927.html
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1) You don't have to satisfy Nyquist; you can undersample if you
want, but then you are throwing away resolution.
2) If you are limited by analog bandwidth, then as you scan along the
fast axis, the PSF of each pixel is elongated along the scan axis by
the low pass filtering in the electronics. 15 us dwell time is
actually 66 kHz, so they are slightly oversampled (Nyquist would be 40
kHz), but the bandwidth on amps is usually 3dB, so there are probably
frequencies well above 20 kHz.
3) Single photon counting extracts more signal per photon, but
typically can only handle extremely low numbers of photons per second.
Operated in analog mode, most PMTs are several orders of magnitude
faster than when in counting mode. If you are not concerned about
illumination power, usually you get a better image per unit time from
analog mode. (however, if you are limited by power, counting is much
better)
Hope that helps.
Mike
On Wed, Oct 26, 2016 at 3:56 PM, Yan, Lu <
[hidden email]> wrote:
> ***** To join, leave or search the confocal microscopy listserv, go to:
>
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>
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