Two-photon microscopy has become a powerful tool in neuroscience as it can image and manipulate neural circuits in vivo with cellular resolution. But in conventional two-photon microscopes, a single laser beam scans regions of interest on the sample within a two-dimensional plane. This serial scanning constrains the temporal resolution of imaging and photostimulation. One way to overcome this limit is to increase the number of beamlets on the sample. Here, we discuss our recent progress on holographic beam multiplexing in two-photon microscopy using spatial light modulators (SLMs). The SLM generates a 3D holographic excitation pattern, targeting different cells on the sample simultaneously. In transparent samples such as zebrafish larva where wide field detection can be used, groups of neurons in 3D volume can be imaged simultaneously, and their fluorescence signals can be recorded with extended depth of view techniques. In scattering samples such as mice cortex which needs laser scanning for imaging, multiple planes can be imaged simultaneously, with the signals from different planes being separated by novel statistical algorithms. Using this approach, we also recorded neural activity across multiplanes in moving Hydra. Besides imaging, 3D optogenetics can be performed. We demonstrate 3D patterned photoactivation of groups of target neurons on mice cortex in vivo, while simultaneously monitoring activity of the neural network. Furthermore, spatial light modulators can switch patterns in high speed, facilitating time-multiplexing. SLM-based two-photon microscopy is thus an all-optical platform to study neural circuits in 3D.
Weijian Yang and Rafael Yuste, "Two-photon holographic imaging and optogenetics of neural circuits (Conference Presentation)," Proc. SPIE 10502, Adaptive Optics and Wavefront Control for Biological Systems IV, 1050215 (Presented at SPIE BiOS: January 29, 2018; Published: 15 March 2018); https://doi.org/10.1117/12.2293559.5752139970001.
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