Re: confocal microscope efficiency measurement

Posted by Jason Swedlow on
URL: http://confocal-microscopy-list.275.s1.nabble.com/confocal-microscope-efficiency-measurement-tp3626645p3643190.html

Hi Francisco-

Great project!

Actually, knowing the number of photons emitted from a fluorescent source (knowing in the sense of having a standard where the exact production of photons is "known") is very difficult.  Not impossible, but difficult.  You can get the efficiencies of the individual elements in the light path, and multiply them together.  This will give you an estimate of the efficiency.  John Murray did this in his chapter in Goldman and Spector's "Live Cell Imaging".

However, you can imagine coming up with a standard sample, and using that to compare different microscopes.  This was done recently:  search Pubmed for PMID: 18045334.  This might give you an idea for a standard sample you might use.

Good luck!

Cheers,

Jason

On Fri, Sep 11, 2009 at 4:42 PM, Francisco Henriques <[hidden email]> wrote:

 Dear all,

 First of all, hello everyone.

 I am new to the mailling list and the microscopy world.
 I'm a portughese student and am currently doing my master thesis at the
Instituto Gulbenkian de Ciência under the guidance of Nuno Moreno.

 We are trying to measure  the light capturing efficiency of a confocal
microscope but aren't sure of the methodology and were hoping you could shed
some light on it.

 The efficiency we want to measure is the fraction of photons emitted from a
light source that is detected in the final image, being: efficiency= np/ne,
where np is the number of detected photons and ne is the photons emitted by a
calibrated light source(LED).

 To calculate np we assumed there was no amplification noise, and used the mean
of the image M and it's variance V.
M=np*g and V=np*g^2.
Being g the average pixel intensity resulting from the detection of one photon,
which is proportional to the gain of the PMT and subsequent elecronics. And np
the average detected photons per pixel.

 Our doubt is in the calculation of ne.
Is each pixel a representation of all the light that reaches the lens during the
pixel dwell time?

 If so, ne should be the number of photons that reach the lens during the dwell
time.

 If not, if each pixel represents only a portion of the light that reaches the
lens during the dwell time, than ne should be a fraction of  the number of
photons that reach the lens during the dwell time.

 I have searched, but I am unable to find any kind of tutorial to help me
understand this concept. If somebody could point me in the direction of a good
source to understand how this should be done, I would be very grateful.

 Many thanks

 Francisco Henriques

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