Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Precisely-driving-several-devices-from-IgorPro-through-National-Instruments-board-possible-tp4907273p4914602.html
Re: photons vs.
photoelectrons?
The recent release of the Photometrics
EMCCD "eVolve" camera which has the ability to output images
with pixel values that correspond to photoelectron counts (instead of
arbitrary digital count units) has me wondering a bit something. The
idea behind this camera, as I understand it, is that having images
reported in terms of photoelectrons instead of counts (ie: an absolute
scale vs a relative scale) will allow a better comparison of image
data between labs around the world, and even for single user comparing
images acquired with a particular microscope from day to day. Seems
reasonable to me and sounds like a pretty good idea. What I'm confused
about is that I've seen some papers in the literature, mainly those
that deal with single-molecule studies, that report image data in
terms of actual photons detected. Is there a difference, and more
importantly, can someone explain to me the advantage of using photon
counts vs. photoelectron counts?
(No commercial interest for
Photometrics)
John Oreopoulos,
BSc,
PhD Candidate
University of
Toronto
Institute For
Biomaterials and Biomedical Engineering
Centre For Studies
in Molecular Imaging
Hi John,
First of all, congratulations to Photometrics! (I think that
other companies may also offer this feature?)
Those of us who have been following developments in
phototdetectors have long recommended this approach although the
EM-CCD really made it essential.
Photoelectron counts are the best you can do. You can't actually
count photons until they have been turned into photoelectrons. The
quantum efficiency is the fraction of photons that are converted into
photoelectrons (and subsequently detected). It is often a strong
function of wavelength.
There are many advantages of this sort of calibration. Although,
as others suggest, it facilitates inter-lab calibrations etc, I think
that the main advantage is that is allows the operator to make a good
estimate of Poisson Noise and without this, any flirtation with
quantitative fluorescence is impossible.
Poisson noise defines the uncertainty of the photon signal
intensity as the sqrt of the number of quantum events
(photoelectrons), not the sqrt of some number stored in the computer
that is only proportional to the signal intensity.
Finally, it allows you to greatly compress the data without loss.
In the early days, NASA used to convert their CCD signal into
electrons and then take the sqrt of this number before sending it back
home (through a very slow, noisy signal channel). It halves the number
of bits needed.
Now, if we could just get the confocal manufacturers to do the
same. Admittedly determining the proper calibration is much harder to
do with PMTs than CCDs, but it is not at all impossible.
Cheers,
JIm Pawley
--
********************************************************************
*************
Prof. James B.
Pawley,
Ph. 608-263-3147
Room 223, Zoology Research
Building,
FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706
[hidden email]
3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver
Canada
Info:
http://www.3dcourse.ubc.ca/
Applications
still being accepted
"If it ain't
diffraction, it must be statistics." Anon.