A NOVEL TWO-PHOTON 3D RANDOM-ACCESS AOD-BASED MICROSCOPE WITH TEMPORAL FOCUSING FOR IMPROVED OPTICAL SECTIONING

The two-photon 3D Custom Access Serial Holography (3D-CASH) microscope, based on the control of Acousto-Optic Deflectors (AODs), has enabled volumetric neuronal activity recordings in the kHz range in head-fixed behaving mice. Although the use of AOD-based holographic beam shaping to enlarge excitation volume efficiently improves fluorescent signals and mitigates in vivo motion-induced recording artefacts, it also introduces interference patterns (“hot spots”) away from the excitation pattern, that may compromise the specificity of the detected neuronal signals in dense neuronal population labelling. In this study, we aim to enhance optical sectioning in the 3D-CASH microscope by implementing temporal focusing (TF). We demonstrated both numerically and experimentally the feasibility of combining TF with AODs despite inherent spatio-temporal pulse distortions generated by AODs. Using numerical simulations based on a ray-tracing method (Kostenbauder matrix theory), we showed that defocus and group delay dispersion can be successfully corrected at the center of the field-of-view by adding an Acousto-Optic Modulator (AOM) before the AOD in the TF configuration. Based on the development of a modular 3D-CASH set-up with TF, we experimentally confirmed that adding the AOM is critical to recover near transform-limited pulses at the focal plane. Finally, we demonstrated that hot spots induced by holography could be efficiently suppressed using TF, thus improving optical sectioning. By removing the background contribution to the total collected fluorescence, introducing TF to the 3D-CASH set-up should considerably enhance the specificity and the signal-to-background ratio of activity recordings, paving the way for activity recordings in larger neuronal networks.