Abstract
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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Citation
Benjamin Brenner, Wei-Hong Yeo, Youngseop Lee, Junghun Kweon, Cheng Sun, and Hao F. Zhang. 2025. “Implementation and calibration of spectroscopic single-molecule localization microscopy.” BMC Methods. https://doi.org/10.1186/s44330-025-00022-x.
@article{Brenner2025,
author = {Benjamin Brenner and Wei-Hong Yeo and Youngseop Lee and Junghun Kweon and Cheng Sun and Hao F. Zhang},
doi = {10.1186/s44330-025-00022-x},
journal = {BMC Methods},
title = {Implementation and calibration of spectroscopic single-molecule localization microscopy},
year = {2025}}