A fallacy in the interpretation of fluorescence decay parameters

Abstract It is shown that single-photon-counting fluorescence decay data fit to a two-component decay law yielding satisfacory fit statistics and random residuals can conceal underlying sets of lifetimes with a variety of intensity distribution functions of remarkable width and diversity of shape. Only by collecting data to a large number of counts per channel (5 × 10 4 to 5 × 10 5 in the peak channel) with equipment free of common faults can one see even a hint of the presence of an underlying complex distribution of decay times. This conclusion has important and disturbing implications in studies where fluorescent molecules can emit under a variety of environmental conditions such as in proteins, on surfaces, in micelles and membranes, in cold beams clustered with other molecules, in polymers, etc.