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    This study enhances two-photon excitation fluorescence (TPEF) microscopy by broadening the excitation source bandwidth using photonic crystal fiber. This allows for faster spectral window tuning, improving multiplexing capabilities for biological imaging.

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    Area of Science:

    • Biophotonics
    • Microscopy
    • Fluorescence Imaging

    Background:

    • Two-photon excitation fluorescence (TPEF) microscopy is crucial in biological research.
    • Limited multiplexing capability due to narrow light source spectral bandwidth hinders TPEF applications.

    Purpose of the Study:

    • To broaden the excitation source bandwidth in TPEF microscopy.
    • To enable fast spectral window tuning for enhanced multiplexing.

    Main Methods:

    • Utilized photonic crystal fiber to broaden the excitation source.
    • Employed a spatial light modulator as a programmable diffraction grating for spectral window tuning.
    • Incorporated a grating pair for dispersion compensation to enhance excitation efficiency.

    Main Results:

    • Achieved a broad excitation spectrum with fast spectral window tuning (tens of milliseconds).
    • Successfully demonstrated the method by imaging live B16 cells labeled with four spectrally overlapped fluorescent proteins.

    Conclusions:

    • The developed method significantly enhances the multiplexing capability of TPEF microscopy.
    • Broadened spectral bandwidth and programmable tuning offer advanced possibilities for live-cell imaging and biological studies.