Comparison of approaches to the instrumental response function in fluorescence decay measurements

Deconvolution of pulse fluorometry data requires knowledge of the instrumental response, which is not directly observable in some circumstances. Various procedures for approaching the instrumental response function were evaluated for nanosecond fluorescence decay data analyzed by nonlinear least squares, including the commonly used time shift correction and several reference fluorophore methods. A new reference fluorophore technique using a Monte Carlo convolution is introduced and tested. The correction for scattered light in several reference techniques is also presented. The random convolution and one other reference fluorophore method consistently gave superior results over a wide range of experimental conditions.