Tetra-color superresolution microscopy based on excitation spectral demixing

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.