Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/averaging-vs-accumulation-for-noise-reduction-is-there-a-difference-tp6483751p6485603.html
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Hi Stan,
Also depends on the fluorophore(s), probably with respect to both
photobleaching (destruction) and driving to triplet state(s) - re T-Rex
mentioned in previous reply.
On our Leica SP5, Jonathan Boyd of Leica showed me a comparison of:
* resonant scanner (8000 Hz), 10 frame average
vs
* standard scanner, 800 Hz, single scan
PMT gain (and offset), field of view, number of pixels in image, were
the same. Sorry, I forget what specimen (could have been Convallaria stem).
Resonant scanner mode was clearly brighter signal.
I suppose it is possible that Leica's digitizer was doing things
differently in the two modes.
On our Zeiss LSM510, different scan speeds have some impact on
intensity. The Zeiss field service engineer told me they try to
calibrate all speeds to produce the same intensity. Our 510 produces
brightest signal - but also noisiest - at fastest scan speed (i.e. on
Chroma fluorescent slides where I assume items in line one above are not
an issue).
Upshot, your signal may vary for reasons beyond your expectations.
Enjoy,
George
On 6/16/2011 12:26 PM, Stanislav Vitha wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hallo,
> this is a very basic question, but I cannot figure this out from what I have
> been reading, so a simple explanation for a non-physicist would be much
> appreciated:
>
> Is there a real difference in the improvement of the signal to noise ratio
> between frame averaging (or accumulation) and longer dwell times (slower
> scan) for a point-scanning confocal witrh a PMT detector?
>
> For instance, using single point scanning confocal, 12-bit acquisition.
>
> a) averaging (or accumulating) 5 frames, 4 microseconds per pixel
> b) acquiring a single frame, 20 microseconds per pixel
>
> Assumptions:
> no saturation of the detector;
> stable environmental conditions, no focus drift, etc
>
> Would it matter (for the dfference between the two scenarios) if it was analog
> detection or photon counting detection?
>
> I will run this little test later, but I am curious what you think.
>
> I thought that at least for the photon counting mode, the two important
> factors would be the dark counts and the total number of counts detected, so
> whether it is acquired in one scan or in 5 scans, it should be the same. My
> camera expert here insists that the averaging scheme will give better noise
> suppression.
>
> Thanks!
>
>
> Stan Vitha
>
> Microscopy and Imaging Center
> Texas A&M University
>
>
--
George McNamara, PhD
Analytical Imaging Core Facility
University of Miami