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    We developed spectroscopic single-molecule localization microscopy (sSMLM) to simultaneously capture spatial and spectral data from single molecules. This technique enables high-throughput dye characterization and multicolor super-resolution imaging with a compact design.

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    Area of Science:

    • Optical microscopy
    • Spectroscopy
    • Biophysics

    Background:

    • Single-molecule localization microscopy (SMLM) provides super-resolution imaging.
    • Characterizing spectral properties of fluorophores is crucial for multicolor imaging.
    • Existing methods may lack throughput or simultaneous spectral information.

    Purpose of the Study:

    • To develop a novel spectroscopic single-molecule localization microscopy (sSMLM) technique.
    • To characterize spectral heterogeneities of far-red emitting dyes.
    • To enable simultaneous multicolor super-resolution imaging.

    Main Methods:

    • Developed transmission diffraction grating-based sSMLM.
    • Collected spatial and spectral information of single-molecule blinking events concurrently.
    • Investigated spectral dispersion effects on fluorophore identification.

    Main Results:

    • Characterized spectral heterogeneities of multiple far-red dyes in a high-throughput manner.
    • Demonstrated simultaneous three-color super-resolution imaging in fixed cells.
    • Achieved imaging with a single objective lens and a low photon budget.

    Conclusions:

    • Grating-based sSMLM offers a compact optical design for spectroscopic analysis.
    • The technique can be integrated with conventional localization microscopy.
    • sSMLM enables efficient multicolor super-resolution imaging and dye characterization.