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    This study introduces an improved parallelized image scanning microscopy (ISM) technique for faster super-resolution (SR) 3D imaging. The enhanced method achieves sub-second SR volumetric imaging, enabling real-time observation of live biological samples.

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    Area of Science:

    • Biomedical Imaging
    • Microscopy
    • Cell Biology

    Background:

    • Super-resolution (SR) microscopy is vital for biological research.
    • Volumetric imaging of thick samples with SR microscopy is slow and complex.

    Purpose of the Study:

    • To develop an enhanced parallelized image scanning microscopy (ISM) system for rapid SR volumetric imaging.
    • To overcome speed and complexity limitations in SR imaging of biological samples.

    Main Methods:

    • Incorporated a pinhole illumination system to create a needle-like beam, extending the depth of focus.
    • Utilized a multiplane prism for synchronized capture of multifocal signals from eight sample planes.
    • Improved reconstruction algorithms to reduce raw frame requirements.

    Main Results:

    • Achieved sub-second SR imaging of a 42 × 37 × 1.9 μm³ volume.
    • Maintained axial imaging synchronization during rapid volumetric acquisition.
    • Successfully demonstrated real-time SR volumetric imaging on live cell samples.

    Conclusions:

    • The enhanced parallelized ISM system significantly accelerates SR volumetric imaging.
    • This breakthrough enables real-time SR imaging of dynamic biological processes in live cells.
    • The system shows great potential for advancing biomedical imaging applications.