Abstract

We developed transmission diffraction grating-based spectroscopic single-molecule localization microscopy (sSMLM) to collect the spatial and spectral information of single-molecule blinking events concurrently. We characterized the spectral heterogeneities of multiple far-red emitting dyes in a high-throughput manner using sSMLM. We also investigated the influence of spectral dispersion on the single-molecule identification performance of fluorophores with large spectral overlapping. The careful tuning of spectral dispersion in grating-based sSMLM permitted simultaneous three-color super-resolution imaging in fixed cells with a single objective lens at a relatively low photon budget. Our sSMLM has a compact optical design and can be integrated with conventional localization microscopy to provide add-on spectroscopic analysis capability.

© 2019 Optical Society of America

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