The time‐resolved fluorometry can be used to probe the rather complex biological macromolecule structure in solution. The interrelationship between structure variability, structure dynamics in the nanosecond time scale, and biological function are studied in measuring spectroscopic and rotational dynamics parameters of the fluorescent moiety in biosystems. The time‐correlated single photon counting technique, using synchrotron radiation as an excitation pulse, has been used to measure the decays of polarized components of fluorescence. This technique has a very high sensitivity so that extremely low quantum yield or very dilute material can be measured. Furthermore, the statistical errors on the data obeys Poisson statistics. A description of the successful analysis of polarized pulse‐fluorescence lifetime distributions provide insights into the local environment of fluorophores and the conformational heterogeneity and intermolecular interactions of biosystem. We also demonstrate that MEM is able to extract full and correct description of conformational dynamics behaviour, in term of rotational correlation time distributions of an heterogeneous fluorescent emission. However, it is shown that the data still leave some ambiguity in the allowable distributions and that the MEM results reflect this ambiguity. Several applications on single tryptophan proteins are presented.

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