Re: photons vs. photoelectrons?

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

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  [phone]
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.