Re: photons vs. photoelectrons?
Posted by
Andreas Bruckbauer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Precisely-driving-several-devices-from-IgorPro-through-National-Instruments-board-possible-tp4907273p4964984.html
Hi Mark,
sorry if anyone is bored by this topic now, but i think counting single photon events after thresholding is a bad idea because the result depends very much on the threshold settings, the signal for one photon and read out noise is not well enough separated, i would rather trust the established methods.
best wishes
Andreas
-----Original Message-----
From: Mark Cannell <
[hidden email]>
To:
[hidden email]
Sent: Fri, 23 Apr 2010 22:37
Subject: Re: photons vs. photoelectrons?
Hi Andreas
As I said at the beginning, there are very few cases where actual photon
numbers are needed, but it adds a veneer of precision/expertise to put
out an image "calibrated" in photoelectrons. Now I don't mind that, but
if it's to be done that way I would like it to be correct/honest. I hope
you can see my point.
As in other areas, the purpose of calibration is to allow reference to
others. But in my experience it is hard to do a good calibration of most
complex measurements so it's better if a result can be expressed in
terms of a change... The only cases I can think of where actual quantum
numbers are needed are for some statistical tests or fitting to theory.
The trouble with EMCCD is that the multiplicative noise reduces the S/N
so it's as if you actually got about half the number of photons. (So,
if you are in a regime where your signal for the exposure time is much
greater than the read noise you should not use an EMCCD. While most
EMCCDs also allow you not to use the EM register, the read out amplifier
for the CCD shift register is also very noisy by good CCD standards. )
But With an EMCCD, 'accurate' calibration is actually easier when you
can detect a signal with mean signal per pixel <<1 phot. Now when you
_count_ (by thresholding) events you have removed the problem of
multiplicative noise so when you take the average signal intensity
(minus dark frames of course) you know how many photoelectron events are
associated with it. As far as I know, no software/camera does this -but
you can.
Cheers
Andreas Bruckbauer wrote:
> I have a few questions regarding this:
>
> - What is the point in knowing how many photoelectrons have been
> detected when photons get lost all the way through the microscope and
> the number of photons depends on other parameters like illumination
> intensity and environment of the dye?
>
> - Mark, you seem to be so confident about your way to calibrate the
> camera, how do you do it?
>
> - The method with dark frames and flats is described by Gosh and Webb
> in Biophysical Journal Volume 66 May 1994 1301-1318, they write: "this
> provides a lower
> boundary for the actual number of photons detected, because other
> noise contributions with similar square-root dependencies may exist."
>
> - Has anyone actually compared the results of these calibration with a
> result of an illumination of a known number of photons?
>
> best wishes
>
> Andreas
>
>
>