Implementation and calibration of spectroscopic single-molecule localization microscopy

Spectroscopic single-molecule localization microscopy allows multi-color super-resolution images with high spectral sensitivity. While its profile has been steadily growing, it is still under-utilized in biology labs, and the complexity of adoption for existing is prohibitive. In this protocol, we provide essential information for researchers to implement sSMLM in a laboratory setting. We describe how to assemble and optically align the illumination and detection paths of a 3D dual-wedge prism-based sSMLM instrument. We provide detailed step-by-step instructions on 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. By demonstrating an implementation of spectroscopic single-molecule localization microscopy that is simple to implement with commercial microscope systems using freely distributed hardware and by introducing new calibration and image processing user interfaces, we hope to remove some of the barriers to entry that biological labs face when entering the spectroscopic super-resolution field. Ultimately, this protocol will allow users to achieve multi-color super-resolution imaging using a single excitation wavelength.