Re: Nyquist theorem and DAQ sampling rate, pixel rate, laser rep. rate, and detector/amp bandwidth

Posted by Lu Yan 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-tp7585926p7585929.html

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Hi Mike,

Thanks for your reply. I am sorry for the mis-calculation, I meant to write 0.0667MHz... :J

Anyway, for the second comment you put, would it be 'undersanpled' instead of 'oversampled' if your pixel rate is 66kHz and amp 20 kHz? Supposed that you have a line sample (1x8) with intensity variation pixel by pixel, e.g. something like this: 'o x o x o x o x', where o denotes fluorophore on-site, and x means no signal. If you scan at 66 kHz, then would you be able to catch the second pixel 'x' and probably the third pixel 'o' because your pmt output is only 20 kHz, i.e. more than three times slower than pixel scanning rate? So effectively, will you not probably get an image something like ' o o o x x x o o'? Should the Nyquist pixel rate be 10 kHz?

Thanks,
Lu


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Giacomelli
Sent: Wednesday, October 26, 2016 4:30 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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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:
> 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