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    This study introduces a new method to accurately size nanoparticles using single-molecule localization microscopy (SMLM). The technique corrects errors in sizing small vesicles and extracellular vesicles (EVs), improving accuracy for nanostructures below 100 nm.

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    Area of Science:

    • Biophysics
    • Nanotechnology
    • Microscopy

    Background:

    • Single-molecule localization microscopy (SMLM) enables super-resolution imaging of nanostructures.
    • Accurate sizing of nanoparticles smaller than 100 nm using SMLM is challenging due to localization errors.

    Purpose of the Study:

    • To develop and validate a method for correcting localization errors in SMLM-based nanoparticle sizing.
    • To compare sizing accuracy using different vesicle labeling schemes.

    Main Methods:

    • Derived a size correction equation using mean approximation theory and full width at half-maximum (FWHM).
    • Validated the method by sizing nanobeads using dSTORM SMLM and comparing with transmission electron microscopy (TEM).
    • Applied the corrected method to size seminal extracellular vesicles (EVs) labeled with membrane dyes and antibodies.

    Main Results:

    • The error-correction method significantly improved the accuracy of SMLM sizing for nanobeads smaller than 50 nm compared to TEM.
    • Error correction reduced size overestimation for 40 nm, 30 nm, and 20 nm nanobeads by 8%, 35%, and 51%, respectively.
    • Sizing of seminal EVs showed smaller diameters after error correction, with differences varying based on labeling method (membrane dye vs. antibody).

    Conclusions:

    • The developed error-correction method enhances the accuracy of SMLM for sizing small vesicles and nanoparticles.
    • Compact membrane labeling schemes, combined with error correction, minimize overestimation of vesicle sizes by SMLM.
    • This computationally inexpensive method is compatible with various SMLM techniques.