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