Abstract

Spatial resolution of stimulated emission depletion (STED) microscopy varies with sample labeling techniques and microscope components, e.g., lasers, lenses, and photodetectors. Fluctuations in the intensity of the depletion laser decrease achievable resolution in STED microscopy; the stronger the fluctuations, the higher the average intensity needed to achieve a given resolution. This phenomenon is encountered in every STED measurement. However, a theoretical framework that evaluates the effect of intensity fluctuations on spatial resolution is lacking. This paper presents an analytical formulation based on a stochastic model that characterizes the impact of the laser fluctuations and correlation time on the depletion efficiency in continuous-wave (CW) STED microscopy. We compared analytical results with simulations using a wide range of intensity noise conditions and found a high degree of agreement. The stochastic model used considers a colored noise distribution for the laser intensity fluctuations. Simple analytical expressions were obtained in the limit of small and large fluctuations’ correlation time. These expressions fitted very well the available experimental data. Finally, this work offers a starting point to model other laser noise effects in various microscopy implementations.

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