Re: FRET - PMT Spectral Sensitivity

Posted by yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/FRET-PMT-Spectral-Sensitivity-tp4720210p4720368.html

Hi Pablo,

 

My name is Yuansheng Sun, working for Dr. Periasamy in the KCCI at UVA. I am currently maintaining the PFRET software.

 

If you use a CCD camera, which should usually give the same quantum efficiency for the eCFP and eYFP emission wavelengths, then you just leave both sensitivities as 1:1. If the PMT detectors are used, the sensitivities for eCFP and eYFP could be different, depending on the PMTs. You should be able to get the PMT quantum efficiency graph from where you bought the system or the PMT. Based on the graph, you can obtain the sensitivities at the peak emission wavelengths of eCFP and eYFP.

 

Details on why we need to consider the difference between the detector sensitivities for donor and acceptor are explained in Chapter 7 of Molecular Imaging, FRET Microscopy and Spectroscopy, edited by Ammasi Periasamy and Richard N. Day.

 

sheng

 

On Thu, Mar 11, 2010 at 10:17 PM, Pablo German <[hidden email]> wrote:

Hi,

I'm analysing FRET images using the pFRET plugin of ImageJ developed
at Universit of Virginia.

When setting the parameters it asks me for the PMT Spectral
Sensitivity for donor and acceptor. I'm using eCFP and eYFP. Where can
I find these values? Is it important?

Regards,
Pablo

--
Pablo German
PhD Candidate

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