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Stan,
I suggest that you adjust voltages and offsets on
your PMTs, test the variations of dark noise
counts with PMT voltage, and make sure that you
are operating on a linear portion of PMT gain
range. The large Max counts you are seeing is a
bit strange. Cosmic rays could generate some
anomalies but given that the STDs are
consistently more than twice the expected values
some systematic problem is more likely. What I
find the most eye catching is your getting ~ 10
dark counts per 200 usec. dwell time. That
implies your PMT(1&2) are generating 50,000
counts per second (-> 250,000 per sec. for PMT
3!). That's likely to be about 60-200x higher
than what one gets with a typical 2 inch PMT, a
discrepancy worthy of examination. If the gain
settings are too high you might end up with a lot
of spurious counts. I would certainly check with
manufacturer to get the specs. of the PMTs.
Another factor that comes to mind is cooling of
your detectors. The noise from PMTs can be helped
a lot by cooling. If PMTs are so equipped, I 'd
check to make sure that the cooling is working.
Going from room temp. down to -20°C can reduce
thermionic emission by greater than a factor of a
hundred.
There was another consideration mentioned in your
posts that you could get about 200 counts per
pixel with a 2 usec dwell in your test specimen.
"100 x 2 us dwell time, Sum of frames
area (pixels) mean STDEV signal-to-background
specimen 20812 185.28 33.97 14.73"
This is means you are getting about 1 count per
10 nsec. Although this depends on the PMT, a
common pulse width is in the neighborhood of 5
nsec. This is only a rule of thumb but you need
to be close to 1/10 of this flux to avoid
coincidence counting error. Photon counting rates
higher than 40 counts per 2 usec. will
unavoidably start losing counts, i.e., show signs
of saturation. There are some tricks that can
help correct for this error or at least make a
reasonable estimate (e.g., correlate count(s)
with size of the current pulse per detected
photon; overlapping pulses will generate ~ twice
total charge of single photon event). Actually,
from your laser power table, it does appear that
your are showing some saturation effects when
comparing the values from 1% power and higher.
It is also interesting that in virtually all your
measurements, your STDev are very consistently
2.5-2.6 times the expected Poisson number. In
fact, this error is so consistent that it looks
systematic:
Laser Power STDEV/sqrt(mean counts)
0.1% 2.49
0.5% 2.49
1% 2.46
2% 2.49
4% 2.53
8% 2.51
16% 2.55
32% 2.55
I wonder if your system&A/D converter are
employing an offset and performing some kind of
scaling? Even your dark counts look like a factor
of 2.5 is being applied. I have to ask, is this
really photon counting? "Hybrid photon counting"
makes me think it is not counting photons but
estimating them. Sorry, haven't used the FV1000
or I might be able to provide an answer?
As for accumulating versus using very long dwell
times, if true photon counting is being used, the
statistics are straight forward, averaging and
accumulating are equivalent. The noise should
follow the sqrt rule. Long dwell times especially
at high power will enhance bleaching and
photodynamic damage, but to a first order one can
imagine that most decay processes will still sum
their rates exponentially so that noise will
track the accumulated signal the via sqrt law.
Let us know, Stan, how things turn out and thanks
for taking the time to do the measurements.
Mario
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>*****
>
>I have a little update.
>Some commented that the noise in my photon counting experiment is higher
>than expected from Poisson distribution.
>
>I checked the dark counts on the PMTs (kept in the dark overnight, all light
>blocked, lasers off during the scan)
>512 x 512 pixels scan, 200 us dwell time.
>
>PMT 1 mean = 9.80 Min = 0 Max = 101 STDEV = 7.98
>PMT 2 mean = 8.75 Min = 0 Max = 126 STDEV = 8.68
>PMT 3 mean = 49.69 Min = 0 Max = 181 STDEV = 18.18
>
>PMT1 was the one used in the previous test.
>
>I expected that the dark count would have Poisson distribution, but again the
>STDEV is more than twice the SQRT(mean).
>
>It could be a questtion of the threshold set for photon counting, but I think
>only gods on Olympus would know..
>
>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
>
>On Tue, 28 Jun 2011 11:33:13 -0400, Chen, De (NIH/NCI) [C]
><[log in to unmask]> wrote:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>*****
>>
>>It seems, in this experiment, the count rate is way too high to see the shot
>noise effect. The Poissonian noise with
>uncertainty 1/sqrt(N), longer dowelling
>time will increase S/N. When signal is very weak, longer dowelling time will
>help.
>>
>>________________________________________
>>From: James Pawley [[log in to unmask]]
>>Sent: Monday, June 27, 2011 9:10 PM
>>To: [log in to unmask]
>>Subject: Re: averaging vs. accumulation for noise reduction - is there a
>difference?
>>>Also, I note that your STDEV is NOT equal to the sqr root of the
>>mean. I would guess that they have merely calibrated their PMT system
>>so that the number in the memory is about equal to the number of
>>single PE pulses that occurred in the pixel time.
>>
>>JP
>>
--
________________________________________________________________________________
Mario M. Moronne, Ph.D.
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