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Oops--I accidentally inverted the equation. It should be QY=QYstandard*(F/Fstandard) *(Astandard/A)*(n^2/nstandard^2).
For quantum yield standards, see Magde et al photochemistry and photobiology 2002 vol 75 p 327. It gives a thorough study for fluorescein and rhodamine 6G. Other wavelengths are a bit more difficult. Lakowicz principles of fluorescence spectroscopy gives more details and a few standards from the literature.
That's a pretty neat method! It avoids having to worry about figuring out photon flux by calibrating against a known. Where would you get trustworthy QE calibration standards though?
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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
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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
>
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> Optical Imaging Techniques in Cell Biology
> by Guy Cox CRC Press / Taylor & Francis
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