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    Area of Science:

    • Biomedical Optics
    • Microscopy
    • Biophysics

    Background:

    • Image scanning microscopy (ISM) offers optical sectioning capabilities.
    • Traditional 3D microscopy techniques face challenges with penetration depth and resolution in scattering biological tissues.
    • Two-photon excitation provides inherent depth advantages due to its nonlinear nature and longer excitation wavelengths.

    Purpose of the Study:

    • To present a novel two-photon fluorescence image scanning microscopy (ISM) system enabling 3D imaging within a single 2D scan.
    • To demonstrate the capability of this system for imaging thicker biological samples with improved resolution and penetration depth.
    • To combine holographic multispot excitation with advanced detection schemes for enhanced 3D imaging performance.

    Main Methods:

    • Engineered excitation using a holographic multispot array of focused femtosecond pulses.
    • High-efficiency single-helix point-spread-function (PSF) phase mask for detection.
    • Camera detection coupled with a multiview reconstruction algorithm for volumetric data acquisition.
    • Utilized nonlinear two-photon excitation process for improved optical sectioning.

    Main Results:

    • Achieved 3D volumetric imaging of biological samples within a single 2D scan.
    • Demonstrated imaging over a depth of field exceeding 1500 nm.
    • Obtained an axial resolution better than 400 nm.
    • Showcased improved penetration depth in scattering samples due to longer wavelengths and nonlinear excitation.

    Conclusions:

    • The developed two-photon fluorescence ISM system significantly enhances 3D imaging capabilities for biological samples.
    • The combination of advanced excitation and detection strategies extends the performance of 3D ISM, particularly for thicker and scattering specimens.
    • This method offers a promising approach for high-resolution, deep-tissue imaging in biological research.