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    Area of Science:

    • Neuroscience
    • Biophysics
    • Optical Engineering

    Background:

    • Recording 3D neuronal activity with cellular resolution, high SNR, and millisecond temporal resolution is challenging.
    • Random-access two-photon microscopy using acousto-optic deflectors (AODs) offers high sampling rates but suffers from background contamination.
    • Holographically shaped point spread functions (PSFs) in AOD systems increase background noise compared to diffraction-limited PSFs.

    Purpose of the Study:

    • To overcome background contamination in AOD-based two-photon microscopy.
    • To improve SNR and enable high-resolution 3D neuronal activity recording.
    • To develop advanced excitation patterns for in vivo imaging.

    Main Methods:

    • Implemented an AOD scanning system integrating temporal focusing.
    • Compensated for spatiotemporal distortions using an acousto-optic modulator.
    • Designed extended excitation patterns by combining temporal focusing and holographic wavefront shaping.

    Main Results:

    • Successfully superimposed spatial and temporal foci across the field of view.
    • Generated complex, extended two-photon excitation patterns.
    • Achieved significantly improved background rejection compared to 2D holographic patterns.

    Conclusions:

    • The new AOD system with integrated temporal focusing enhances background rejection.
    • This technique offers improved SNR for in vivo neuronal recordings in dense samples.
    • The method holds promise for advanced neuroscience research requiring high-resolution imaging.