http://confocal-microscopy-list.275.s1.nabble.com/Nyquist-theorem-and-DAQ-sampling-rate-pixel-rate-laser-rep-rate-and-detector-amp-bandwidth-tp7585926p7585935.html
Chameleon Ultra or the their Spectra-Physics equivalent. These are run
mentioned for the paper you read), so one pulse every 12.5 nanoseconds.
to minimize "pile up" (which reminds me of Houston traffic accidents). I
p.s. Becker&Hickl also discouraged the use of resonant scanner mode for
The SP5 had 3 standard Leica PMTs and two "FLIM" PMTs. The SP5 was
purchased ~2005, so before HyD or HyD-FLIM detectors. It also Leica
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> Hi Mike,
>
> Thanks for your explanation. I understood that if the sampling rate is higher than twice the analog bandwidth (20kHz), because it simply means sampling more than two points per analog pulse. But in the example, as I understood, the sampling rate (set in the DAQ) is >1MHz per channel (obviously fulfill the 'oversampling' condition), but the pixel rate is 66 kHz, in this case, should I think of 'undersampled' or 'oversampled'?
>
> For the detector, can I interpret the analog bandwidth 20 kHz in this way (in the time domain):
>
> (1) The detector receives fluorescence photons and generate the photocurrent pulse with a raise time and fall time. The output signal pulse (the first pulse) has long tail (probably longer than 0.05 ms?) so that any other pulses coming in within 1/20kHz=0.05ms time window after the first pulse will not be detected or will be attenuated accordingly.
>
> (2) Therefore, if the first pulse is coming from a pixel (see, pixel #1), and the laser quickly moves (quicker than 0.05 ms, e.g. 30 us) to pixel #2 where there is no fluorophore. Now, the DAQ channel samples the detector again, and assigns the signal strength to pixel #2. Since the detector has 20 kHz bandwidth, if (1) holds, then would the channel get some value due to the long tail of the first pulse (coming from previous pixel)? If this is true, then the image is not the correct representation of the object, i.e. pixel #2 actually does not have fluorophores but is showed up in the image as if there are some.
>
> Thanks,
> Lu
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Michael Giacomelli
> Sent: Wednesday, October 26, 2016 5:40 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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> Hi Lu,
>
> No, if the sampling rate (66 khz) is higher than twice the analog bandwidth (20 khz), then the image is oversampled (at least assuming the optical resolution is much higher than the analog bandwidth limit). However, since the analog bandwidth in this case is probably just the 3dB point, the image will contain some information above 20khz, although it will be attenuated.
>
> In your example, it depends on how rapidly you are scanning and what your optical resolution is. If you really mean 15 us per point, then your pattern frequency is 1/30us = 33.333Khz, which is a little bit higher than your 3dB point. Many detectors roll off 6dB per factor of two past the 3dB point, so you would probably be attenuated about 7-8dB. If the points you are imaging are bright, you would still see them.
>
> Mike
>
>
> On Wed, Oct 26, 2016 at 5:10 PM, Yan, Lu <
[hidden email]> wrote:
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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:
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>>>
>>> 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