Computational modeling of stimulated emission depletion microscopy in biological cells under one- and two-photon excitation
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The finite-difference time-domain method is used to simulate the propagation of focused beams used for stimulated emission depletion (STED) microscopy as they scatter through layers of biological cells. Depletion beams that facilitate axial and lateral confinement of the fluorescence emission are modeled, and the effective point spread function of the system as a function of focal depth is assessed under one- and two-photon excitation. Results show that the lateral depletion beam retains a well-defined minimum up to the maximum simulation depth of 42 µm. In addition, the relative spatial shift between excitation and de-excitation beam foci is less than 44 nm for all simulated depths. PSF calculations suggest that sub-diffraction imaging is possible beyond the maximum simulated depth, as long as the fluorescence emission is detectable. However, strong attenuation of the fluorescence emission by the axial confinement beam may make this beam unsuitable for sub-diffraction imaging in scattering samples.