New advances in the research of stimulated emission depletion super-resolution microscopy

Abstract

Due to the influence of the diffraction limit, the lateral spatial resolution and axial spatial resolution of traditional optical microscopes are limited to ~200 nm and ~500 nm, respectively. In the past two decades, with the rapid development of high-intensity lasers, high-sensitivity detectors and other optoelectronic devices, there have been reported many super-resolution imaging techniques that bypass the optical diffraction limit with different methods. Among these techniques, stimulated emission depletion microscopy (STED) technology has the advantages of high imaging resolution and fast imaging speed. This technology uses two lasers for imaging, one of which is used to excite fluorescence, and the other donut-shaped depletion laser is used to suppress the emission of fluorescent molecules around the fluorescent spot, in order to reduce the fluorescence point spread function and achieve super resolution Imaging. After recent years of development, the STED system has got great progress no matter from the generation, calibration and scanning of the beam, and the final imaging. In terms of laser source, new laser sources such as continuous wave beams, supercontinuum laser, stimulated Raman scattered laser, and higher-order Bessel beams have appeared; in scanning and calibration, new efficiency technology such as parallel scanning and automatic calibration have also appeared; In imaging, new methods such as time gating and phasor analysis have emerged to improve imaging quality. These new technologies and methods are of great significance to improve the efficiency of STED system construction and imaging. In addition, this paper also focuses on the ways to expand the imaging functions of the STED system. First, for three-dimensional STED imaging, this paper mainly introduces three methods to realize three-dimensional STED imaging by wavefront non-coherent adjustment, 4Pi and structured light illumination methods. Second, for multi-color imaging, this paper introduces several dual-color and multi-color imaging techniques for special dyes. Third, this paper introduces the combination of STED technology with fluorescence correlation spectroscopy technology, cell expansion technology, scanning ion-conductance microscope, photo-activated localization microscopy/stochastic optical reconstruction microscopy and other technologies. Finally, this paper systematically discusses the new research progress of STED technology in recent years, and discusses the future development trend of STED technology.