Temporal Focusing for Enhanced Background Rejection in AOD-Based Two-Photon Serial Holography

The ability to record 3D neuronal activity with cellular resolution, high signal-to-noise ratio (SNR) and millisecond temporal resolution is a major challenge in neuroscience. One powerful method is random-access two-photon microscopy based on acousto-optic deflectors (AODs), which uses a holographically-shaped point spread function (PSF) scanned in 3D to maximize the sampling rate and SNR. However, this approach suffers from greater background contamination due to the holographically shaped PSF than standard two-photon microscopy with diffraction-limited PSF. To overcome this limitation, we implemented a new version of an AOD scanning system, which integrates temporal focusing. The complex spatiotemporal distortions encountered in this configuration, including a significant group delay dispersion associated with the pulse front tilt generated by the AOD, were compensated for by introducing an acousto-optic modulator before the AOD. We designed extended patterns by combining temporal focusing on one direction and holographic wavefront shaping in the perpendicular axis. Taking advantage of the AOD's ability to shape the wavefront at the same speed as the scan, we were able to accurately superimpose the spatial and temporal foci over the entire field of view. Finally, we generated complex, extended two-photon excitation patterns by combining temporal focusing in one direction and holographic multiplexing in the perpendicular direction. These patterns provide significantly improved background rejection compared to 2D holographic patterns, thus offering promising prospects for in vivo recordings of neuronal activity in dense samples with improved SNR.