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-tp4907273p4951847.html

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

-----Original Message-----
From: Mark Cannell <[hidden email]>
To: [hidden email]
Sent: Fri, 23 Apr 2010 4:07
Subject: Re: photons vs. photoelectrons?

Hi All, 
 
Hate to say I told you so... but you do need to do the calibration yourself . By the way, the method they propose has an important and fundamental assumption .... 
 
I suggest that if Photometrics would like to add/explain the conditions under which their calibration method is justified (and they assume _only_ Poisson noise :-O) on the web page that would be a great help to users. 
 
My tenet (that you need to understand/check what is being done) would seem justified :-) 
 
my last 2c 
 
Mark 
 
 
Deepak Sharma wrote: 
> ******Commercial Interest ******Reply from Photometrics****** 
> 
> I would first like to thank the contributors to this subject matter for 
> their input. > 
> Photometrics would like to provide information and actual data which should 
> help answer the points raised and discussed. This can be found at: 
> 
> http://www.photomet.com/useroutreach/042210.php > 
> We discuss measuring analog gain i.e. the cameras e/ADU conversion factor 
> using a mean-variance analysis, how to calculate electron multiplication 
> gain and the ‘flatness’ or photo-response non-uniformity. We also explain 
> that Rapid-CAL and Quant-View features of the evolve camera utilize a 
> combination of these measurements to provide accurate data integrity. 
> 
> We hope this data and information helps clarify some of the questions being 
> asked and we are always happy to answer any questions you may have. 
> 
> 
> Deepak Sharma, Ph.D. 
> Senior Product Manager 
> Photometrics > 3440 East Britannia Drive 
> Tucson, AZ 85706-5006 
>