Spectroscopic single-molecule localization microscopy (sSMLM) allows multi-color super-resolution images with high spectral sensitivity. In this protocol, we provide essential information for researchers to implement sSMLM in a laboratory setting. We describe how to assemble and align the illumination and detection paths of a 3D dual-wedge prism (DWP)-based sSMLM instrument. We provide detailed step-by-step instructions for performing spectral and axial calibration using fluorescent beads and a nanohole array, respectively. We also discuss using sSMLM to image fluorescently labeled cells and report a new MATLAB package, RainbowSTORM v2, to reconstruct super-resolution 3D images. Further, we present representative images as typical anticipated results for users to validate.
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Brenner et al. (2024) studied this question.
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