Ultrafast high-contrast voltage imaging in freely moving animals

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ABSTRACT We propose to develop a targeted illumination confocal (TICO) microscope to enable high speed, large-scale voltage imaging in the brain. This microscope will be based on the combination of two key strategies. The first strategy is high-speed confocal microscopy based on line scanning. The benefit of confocal microscopy is that out-of-focus background is largely rejected by the use of slit detection. We will supplement this background rejection with the additional strategy of targeted illumination. In this way, we will confine our excitation light only to pre-selected regions of interests (e.g., cell bodies or dendrites), minimizing the generation of out-of-focus background in the first place. These strategies will be implemented with the use of a double-sided scanning mechanism that will enable us to perform fast scanning to fully exploit the ultrafast throughput and remarkably low noise capabilities of state-of-the-art CMOS cameras. By combining the complementary advantages of confocal detection and targeted illumination, we expect to significantly improve the contrast and SNR of our voltage imaging relative to the current methods based on cameras. Our specific aims are to build two dual-region TICO microscopes, one adapted for head-fixed animal imaging, and the other adapted for freely moving animal imaging using a miniature microscope objective coupled by a flexible fiber bundle. Establishing the feasibility of TICO microscopy in freely moving animals would constitute a major breakthrough and will lay the groundwork for future research into the relations between voltage activity and behavior in natural conditions. The ability to image freely moving animals will be indispensable for this goal.