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A rigorous and convenient method of correcting for the wavelength variation of the instrument response function in time correlated photon counting fluorescence decay measurements is described. The method involves convolution of a modified functional form F̃s of the physical model with a reference data set measured under identical conditions as the measurement of the sample. The method is completely general in that an appropriate functional form may be found for any physical model of the excited state decay process. The modified function includes a term which is a Dirac delta function and terms which give the correct decay times and preexponential values in which one is interested. None of the data is altered in any way, permitting correct statistical analysis of the fitting. The method is readily adaptable to standard deconvolution procedures. The paper describes the theory and application of the method together with fluorescence decay results obtained from measurements of a number of different samples including diphenylhexatriene, myoglobin, hemoglobin, 4′, 6-diamidine–2-phenylindole (DAPI), and lysine–trytophan–lysine.

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