In this paper,I consider theoretical models of the decay via photobleaching of a sample of surface-immobilized fluorescent molecules excited by a spatially varying laser intensity profile.I show that,with mild restrictions on the photobleaching mechanism,the fluorescence decay measured in a nonuniform excitation profile is always nonexponential.Under the same conditions,the fluorescence decay can always be approximated by a discrete sum of exponentials.A particular example is given in which a homogeneous population of fluorophores with a single (intensity-dependent) photobleaching lifetime,when illuminated by a Gaussian laser,exhibits power law fluorescence decay at long times.These results indicate that the observation of multiple exponentials in single molecule or ensemble photobleaching lifetime measurements can arise solely as an artifact of a spatially varying laser profile and is not necessarily indicative of heterogeneity in molecular internal states,conformation,or local environment.
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