Tetra-color superresolution microscopy based on excitation spectral demixing

• Published in: Light, science & applications science & applications, 2023-01, Vol.12 (1), p.9-9, Article 9
• Main Authors: Wu, Wanyan, Luo, Shihang, Fan, Chunyan, Yang, Tianjie, Zhang, Shuwen, Meng, Wenxiang, Xu, Tao, Ji, Wei, Gu, Lusheng
• Format: Article
• Language: English
• Subjects: 639/624/1107/328/2238; 639/624/1107/328/2239; Dyes; Fluorescent indicators; Fluorophores; Lasers; Localization; Microscopy; Microwaves; Optical and Electronic Materials; Optical Devices; Optics; Photonics; Physics; Physics and Astronomy; RF and Optical Engineering; Science; Stochasticity
• ISSN: 2047-7538; 2095-5545; 2047-7538
• DOI: 10.1038/s41377-022-01054-6

Multicolor imaging allows protein colocalizations and organelle interactions to be studied in biological research, which is especially important for single-molecule localization microscopy (SMLM). Here, we propose a multicolor method called excitation-resolved stochastic optical reconstruction microscopy (ExR-STORM). The method, which is based on the excitation spectrum of fluorescent dyes, successfully separated four spectrally very close far-red organic fluorophores utilizing three excitation lasers with cross-talk of less than 3%. Dyes that are only 5 nm apart in the emission spectrum were resolved, resulting in negligible chromatic aberrations. This method was extended to three-dimensional (3D) imaging by combining the astigmatic method, providing a powerful tool for resolving 3D morphologies at the nanoscale. A fast switching scheme-based excitation resolving method was proposed for fluorescent molecule identification, which enables tetra-color SMLM imaging