Two-photon (2P) excitation can be combined with phase-modulation approaches, such as computer-generated holography (CGH), to efficiently distribute light into two-dimensional, axially confined, user-defined shapes. Applications include lithography, uncaging, optogenetics and fast functional imaging. However, a linear proportionality between lateral shape area and axial extent degrades axial precision for cases demanding extended lateral patterning.To address this limitation, we previously combined CGH with temporal focusing (TF) to stretch laser pulses outside of the focal plane, which combined with 2P’s nonlinear fluorescence dependence, axially confines fluorescence regardless of lateral extent. However, this configuration restricts nonlinear excitation to a single spatiotemporal focal plane, which is the objective focal plane.
Here we report a novel optical scheme enabling remote axial displacement and simultaneous generation of spatiotemporally focused pattern at multiple planes using two spatial light modulators to independently control transverse- and axial-target light distribution. This approach enabled simultaneous axial translation of single or multiple spatiotemporal focused patterns across the sample volume, while achieving the axial confinement of temporal focusing. We utilized the system's novel capability to dissect the functional connectivity between axially distinct neuronal layers in the mice retina.
Finally, we demonstrated that TF enables robust light propagation trough optically and physiologically diverse neural systems including mice brain, zebrafish larva brain and mice retina.
Valentina Emiliani, "Remote axial displacement of spatiotemporal focused patterns through neural systems (Conference Presentation)," Proc. SPIE 10073, Adaptive Optics and Wavefront Control for Biological Systems III, 100731H (Presented at SPIE BiOS: January 30, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2255823.5380600118001.
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