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bioRxiv · 10.1101/2024.08.15.608177

Implementation and calibration of spectroscopic single-molecule localization microscopy

Brenner, B.·Yeo, W.-H.·Lee, Y.·Kweon, J.·Sun, C.·Zhang, H. F.
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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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Brenner, B., Yeo, W.-H., Lee, Y., Kweon, J., Sun, C., Zhang, H. F.. 2024-08-16. Implementation and calibration of spectroscopic single-molecule localization microscopy. https://doi.org/10.1101/2024.08.15.608177

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