Abstract
Fluorescence microscopy is an important tool for studying cellular physiological functions, and the precise localization of the individual fluorescent molecules is crucial for resolution. In recent years, researchers have proposed various single-molecule localization methods with molecular-level precision. Here, we conducted simulation calculations and feasibility studies on two imaging techniques of minimal emission fluxes (MINFLUX) and RASTer scanning a minimum of light (RASTMIN) for single-photon and multiphoton imaging. Our results indicated that the single-photon excitation wavelength had little influence on the localization precision of MINFLUX and RASTMIN, but multiphoton MINFLUX and RASTMIN significantly improved the center localization precision. However, in multiphoton MINFLUX, the average localization precision was slightly compromised. In contrast, benefiting from the advantages of raster scanning, the average localization precision of multiphoton RASTMIN was superior to that of single-photon RASTMIN. Furthermore, to address the issues of reduced average localization accuracy with multiphoton MINFLUX, we propose an increase in the number of scanning points to enhance the performance of multiphoton MINFLUX imaging.
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