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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?
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>To join, leave or search the confocal microscopy listserv, go to:
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>
>Hallo,
>thanks everybody for their input regarding this topic.
>I have some results from a simple test.
>
>Long dwell time (200 us) seem to produce essentially the same signal to
>background ratio as accumulation of 100 images with 2us dwell times:
>signal:
>
>1 x 200us dwell time:
> area (pixels) mean STDEV signal-to-background
>specimen 20812 181.76 34.44 14.63
>background 20812 12.42 8.99
>
>100 x 2 us dwell time, Sum of frames
> area (pixels) mean STDEV
>signal-to-background
>specimen 20812 185.28 33.97 14.73
>background 20812 12.58 8.97
>
>
>The specimen is a Chroma fluorescent slide occupying half of the
>field of view,
>the other half does not have any sample (= background).
>The Sum was calculated in ImageJ (Image-Stack-Z Project-Sum of Slices).
>
>FYI,
>I also tested for the linearity of the signal versus laser power in the photon
>counring mode to see when the pile-up error would become noticeable.
>Using 10 us dwell time, spectral detector was set to narrow width (2 nm band
>pass)to restrict the photon flux. The signal seems to scale well
>with intensity,
>with just a slight drop off at the highest laser powers (but some of that drop
>off can be due to photobleahching; after acquisition I zoomed out and the
>scanned area showed as a darker rectangle):
>(results from an area of 16756 pixels)
> Specimen background
>laser power (%) mean STDEV mean STDEV
>0.1 9.36 7.62 0.67 2.13
>0.2 11.3 8.42 0.65 2
>0.3 13.26 8.89 0.65 2
>0.4 15.21 9.69 0.67 2.1
>0.5 16.85 10.22 0.68 2.09
>1 45.54 16.63 0.68 2.13
>2 82.51 22.64 0.67 2.15
>4 155.69 31.58 0.66 2.09
>8 297.19 43.26 0.73 2.19
>16 567.71 60.79 0.79 2.32
>32 1049.43 82.74 0.86 2.36
>
>
>So it seems that I can count about 100 photons per microsecond without
>having to worry too much about the pile-up.
Then magic is involved. Or something other than what is normally
called photon counting. Do try and find out what they do.
Bit it isn't photon counting with a PMT. (Because even fast PMTs make
single PE pulses that are several ns long and so you would be dead
for at least 40% of the time at 100 MHz.
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
>Does anybody know how how the
>Olympus's "Hybrid photon counting" works?
>
>Spreadsheed file with more details, graphs are here:
>http://microscopy.tamu.edu/instruments/light-microscopy/fv1000-tests
>
>This folder also contains some of the image files.
>
>
>Sincerely,
>
>Stan Vitha
>Microscopy and Imaging Center
>Texas A&M University
--
****************************************
Prof. James B. Pawley, Phone: 604-822-7801
3D Microscopy of Living Cells: Summer Course CELL: 778-919-3176
Info at: http://www.3dcourse.ubc.ca
"If it isn't diffraction, it is statistics":Microscopist's complaint, Anon.
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