Posted by
Guy Cox-2 on
Jun 17, 2011; 2:32pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/averaging-vs-accumulation-for-noise-reduction-is-there-a-difference-tp6483751p6487321.html
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Kees,
The major source of noise in confocal images is shot noise,
that is the random fluctuation in photon number arriving at a pixel.
There are other sources of noise, of course, but shot noise predominates
(see a certain book edited by one J. Pawley). In any case, in most of
the examples given here, other 'obvious' factors, such as PMT voltage,
were controlled. Prima facie, therefore, the images should be
equivalent. We therefore have to look to other factors, such as the way
the microscope talks to its hardware, or excitation into triplet states.
On a simplistic basis, we might expect that there would be more
overhead - ie dead time - when acquiring an image at high speed rather
than slow, so that a slow scan should win out over averaging. However,
the reported claims go in the opposite direction. I have already
suggested one possible explanation. But the other explanation, that
things are over-excited, seems to be running around (sorry!) and there
is a simple experiment we can do to control for this. Do your averaged
scans, then turn your laser power to exactly 50% of the original value
and average for twice the time. If the results are not identical then
you are saturating your fluorophore and that is the source of the
problem.
It must be at least 20 years since I first pointed out that you
should check for saturation, yet I've never met anyone who does it. In
any experiment you should try reducing the laser power, and check that
the fluorescence decreases proportionally. If it doesn't, you are
saturating and, in simple terms, making a rod for your own back.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]]
On Behalf Of Straatman, Kees R. (Dr.)
Sent: Friday, 17 June 2011 5:53 PM
To:
[hidden email]
Subject: Re: averaging vs. accumulation for noise reduction - is there a
difference?
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Very interesting discussion.
I was just wondering. We talk about S/N. If you acquire a single frame
for longer I would expect you also accumulate more noise in the image,
if you average your signal in each image your sample should be more or
less the same while the noise (random) is averaged. So would you not
expect a better S/N with averaging when all else is equal?
Kees
Senior Experimental Officer
Centre for Core Biotechnology Services
University of Leicester, UK
http://www.le.ac.uk/biochem/microscopy/home.html-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]]
On Behalf Of Stanislav Vitha
Sent: 16 June 2011 17:26
To:
[hidden email]
Subject: averaging vs. accumulation for noise reduction - is there a
difference?
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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
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