Serial filter method for fluorescence evaluation

A method has been developed to obtain the reflective spectral data, β_R(λ), for a fluorescent sample by making a simple modification to a conventional poly-chromatically illuminated spectrophotometer. To use this procedure, a series of sharp cut-off filters are inserted successively into the illumination beam of the instrument to obtain a number of spectral curves from which the complete reflectance curve is constructed piece-wise. The calculation of the complete reflectance curve is done wavelength-by-wavelength, starting at about 30 nm above the peak emission of the sample and proceeding toward the shortest wavelength cut-off filter, selecting the spectral data that are the lowest value. When the data from each filter are assembled, the long wavelength portion above the peak emission is made equal to the unmodified spectrum. Examples are given of the method of computation and comparison with bi-spectral data. The serial filter technique is equally applicable to instruments having either 45/0 or d/8 integrating sphere optical geometry.