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Molecular resolution imaging based on two-color single-molecular localization microscopy (SMLM).

Yuhan Nie, Jie Hu, Siying Zhang

    Optics Express
    |June 14, 2025
    PubMed
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    Molecular Imaging by two-color imaging (MITI) enhances optical resolution to nanometer scale for life science research. This super-resolution microscopy technique enables detailed visualization of molecular interactions and structures previously unresolvable.

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    Area of Science:

    • Life Sciences
    • Biophysics
    • Optical Microscopy

    Background:

    • Single-molecule localization microscopy (SMLM) offers super-resolution but struggles with structures below 10 nm and protein interactions.
    • Existing optical super-resolution methods face limitations in resolving nanoscale biological details.

    Purpose of the Study:

    • To introduce a novel two-color SMLM technique called Molecular Imaging by two-color imaging (MITI).
    • To achieve molecular-level resolution, extending super-resolution capabilities to several nanometers.
    • To demonstrate MITI's utility for studying nanoscale biological structures and protein-protein interactions.

    Main Methods:

    • Development of a two-color single-molecule localization microscopy system (MITI).
    • Application of MITI to samples labeled with organic dyes and fluorescent proteins.
    • Validation using DNA-bound dyes and fusion proteins (ffDronpa-FtsZ-PAmCherry1).

    Main Results:

    • MITI achieves super-resolution down to several nanometers, surpassing previous optical limits.
    • Successfully resolved dyes separated by 2-10 nm on ssDNA.
    • Demonstrated visualization of fused fluorescent proteins (ffDronpa-FtsZ-PAmCherry1), enabling protein interaction studies.

    Conclusions:

    • MITI provides unprecedented molecular-scale resolution in super-resolution microscopy.
    • The technique is versatile, compatible with various labeling strategies (organic dyes, fluorescent proteins).
    • MITI is expected to significantly broaden the applications of molecular-scale super-resolution imaging in life sciences.