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Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells
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Pile-up free fluorescence lifetime imaging with a SPAD-based single pixel camera.

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    This study introduces a novel hardware method for single-pixel imaging (SPI) in fluorescence lifetime imaging (FLIM). It overcomes the pile-up limitation in time-correlated single photon counting (TCSPC), enabling real-time biological monitoring.

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    Area of Science:

    • Optics and Photonics
    • Biomedical Imaging
    • Materials Science

    Background:

    • Single-pixel imaging (SPI) is widely used in fluorescence lifetime imaging (FLIM).
    • Time-correlated single photon counting (TCSPC) offers high temporal resolution but suffers from pile-up, limiting photon count rates to 1-5% of the laser excitation rate.
    • This limitation hinders real-time monitoring of biological phenomena.

    Purpose of the Study:

    • To introduce a hardware-based method to eliminate pile-up in single-pixel camera (SPC) based FLIM.
    • To enable high-fidelity lifetime map acquisition at significantly increased count rates.
    • To facilitate real-time monitoring of biological processes using advanced imaging techniques.

    Main Methods:

    • A novel hardware-based method was applied to a single-pixel camera (SPC).
    • The dead time of the single-photon avalanche diode (SPAD) detector was matched to an integer multiple of the laser period.
    • This approach completely avoids the pile-up phenomenon.

    Main Results:

    • Undistorted and high-fidelity lifetime maps were acquired at count rates up to 40%.
    • This count rate is significantly higher than the limit imposed by the classic pile-up phenomenon.
    • The method remains effective even with computational imaging algorithms.

    Conclusions:

    • The developed hardware method effectively overcomes the pile-up limitation in SPI-FLIM.
    • This breakthrough allows for significantly faster data acquisition and real-time biological monitoring.
    • High-fidelity fluorescence lifetime imaging is achievable at unprecedented count rates.