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In order to measure quantum yields by the reference method, one only needs a defined measurement volume, a standard dye of known absorbance (at the microscope excitation wavelength) and quantum yield, and the absorbance of your sample at the microscope excitation wavelength.  Quantum yield is then defined as QY=QYstandard*(A/Astandard) *(Fstandard/F)*(n^2/nstandard^2).  Here n is the refractive index, F is the measured fluorescence intensity and A is the absorbance at the microscope excitation wavelength.  Note that it is important that the standard and your unknown have similar emission spectra.  Otherwise you will have to correct for the wavelength dependence of your microscope detection efficiency.  Of course, if you have enough sample to measure absorbance, you can also measure fluorescence in a cuvette and then there is no point in going to the microscope.

Jay

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Guy Cox
Sent: Thursday, October 06, 2011 8:44 PM
To: [log in to unmask]
Subject: Quantum yield

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I got a rather left field enquiry today, as to whether there were calibration standards for quantum yield.  It seems that the person wants to measure quantum yield under the microscope.  My immediate response was that this is impossible.  Quantum yield is easy enough to measure in a cuvette but would it be possible in a microscope?  You could make a standard of a known concentration of fluorescein in a cell made by a
spacer under the coverslip, but where do you go from there, if   both
quantum yield and extinction coefficient of the test sample are unknown?

Any bright ideas?

                                          Guy  


 

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon) Australian Centre for Microscopy & Microanalysis, Madsen Building F09, University of Sydney, NSW 2006 

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