All-Optical Neurophysiology Using High-Speed Wide-Area Optical Sectioning

Abstract

During my graduate work, I developed and applied optical and optogenetic methods to map neuronal function across large regions of the brain with single-cell resolution using high-speed optical sectioning. I first introduce an all-optical assay to map neuronal function over large areas of brain tissue. The spectral overlap of optogenetic actuators and reporters challenges their simultaneous use, and optical scattering in brain tissue impedes high resolution fluorescence activity recordings. Spectral crosstalk was minimized by combining an optimized variant, eTsChR, of the most blueshifted channelrhodopsin, with a nuclear-localized red-shifted Ca2+ indicator, H2B-jRGECO1a. Wide-area optically sectioned imaging in tissue was achieved with a structured illumination technique that uses orthogonal codes to encode spatial information, named Hadamard microscopy. The combination of eTsChR and H2B-jRGECO1a with Hadamard microscopy allowed widearea maps of neuronal function spanning cortex and striatum. These tools were applied to probe the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Then I introduce a high-speed imaging extension for wide-area all-optical neurophysiology. Hadamard microscopy achieves optical sectioning via differential modulation of in-focus and out-of-focus contributions to an image. Multiple wide-field camera images are analyzed to create an image of the focal plane. The requirement for multiple camera frames per image entails a loss of temporal resolution compared to conventional wide-field imaging. A new computational structured illumination imaging scheme, compressed Hadamard imaging, achieved simultaneously high spatial and temporal resolution for optical sectioning of 3D samples with low-rank dynamics (e.g. neurons labeled with fluorescent activity reporters). The technique was validated with numerical simulations, and then illustrated with wide-area optically sectioned recordings of membrane voltage dynamics in ex vivo mouse brain tissue and of calcium dynamics in live zebrafish brain. Finally, compressed Hadamard imaging enabled high-speed wide-area all-optical neurophysiology mapping of acute mouse brain slices. The combination of eTsChR with jRGECO allows concurrent stimulation and recording of neuronal activity. Compressed Hadamard microscopy enables high-speed, high resolution optical sectioning in wide-area measurements of densely labeled fluorescent neural activity in scattering brain tissue. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in brain tissue.