Low-power two-color STED microscopy based on phasor plot analysis

Abstract

Stimulated emission depletion (STED) microscopy enables visualization of previously indiscernible subcellular structures in biological cells. However, it is costly and complicated to built a STED microscope system because of the dependence on the depletion laser, especially in multicolor imaging. In addition, inefficient fluorescence inhibition on account of the small stimulated emission cross-section results in a huge demand for the power of the depletion laser, which hinders its application to study living cells. Here we present a method based on phasor plot analysis for achieving two-color STED imaging at a relatively low depletion power, which is implemented in a pulsed-STED microscope system with only a pair of excitation and depletion laser beams. Firstly, two fluorescent dyes with similar spectral characteristics but different lifetimes were selected for two-color imaging. Secondly, a depletion laser with a wavelength closer to the emission maximum was applied to boost the depletion efficiency and reduce the required depletion power. This approach makes two-color STED imaging easier and has the potential to realize multi-color STED super-resolution imaging without the need of additional lasers, thereby offering more convenient and efficient service to researchers.