Today?s knowledge of large-scale neural networks is advancing along two orthogonal directions. Spatial (static, structural) connectomic understanding is achieved through optical and electron microscopy; yielding high spatial resolution with limited or no temporal information. Conversely, electrophysiological methods provide an exceptional temporal understanding of millisecond-order neurodynamic activities in vivo, with restrictions placed on spatial information. Unfortunately, these approaches have historically been rather mutually exclusive and incompatible with each other. This proposal is precisely aimed at breaking the methodological barrier between spatial and temporal observation, through innovative 3D optical scanning concepts which rival the temporal resolution of electrophysiology. The resulting system is ideally suited to imaging genetically expressed voltage- sensitive fluorescent markers. The proposed optical microscope is named TranSIM: Trans-Sheet Illumination Microscope. It is designed to observe brain-wide neurodynamics in model organisms (up to 1 mm3) with ~1 ?m resolution in space, and sub- millisecond resolution in time. Unlike a conventional sheet illumination microscope, which illuminates a single x-y focal plane of the detection objective (on the z-axis), the conceived design forms a light sheet in the transverse direction along the z-axis (y-z plane). This sheet of light is then rapidly scanned along the x-axis as it is imaged. Multiple thin z-slices are then collected simultaneously by spatially multiplexing next-generation large-format sCMOS sensors. Regions of Interest, each with slight depth off-set from the focal plane, will map to a segmented part of the sensor, creating an eight-image focal volume in the time of a single frame. As a result, a thick brain volume can be covered by scanning the focal (x-y) plane (~1 mm2) in one direction (in x) at the frame rate of the sCMOS. The fastest sCMOS cameras are currently being jointly developed and optimized for this purpose. A line confocal readout will be implemented electrically by a rolling shutter mode of the sCMOS sensor to minimize contamination of scattering light. With proper Regions of Interest, a large volume (800 x 80 x 1000 ?m3) can be scanned at 780 volumes / second; 100 times faster than today?s fastest 3D optical microscopy systems. A smaller volume (100 x 80 x 1000 ?m3) can be scanned at an unprecedented rate of 6240 volumes / second, reaching the sub-millisecond temporal resolution of electrophysiological sampling. The illuminative sheet can be formed through the same detective objective for enhanced geometrical flexibility, or by an orthogonal objective (as in conventional horizontal sheet illumination) to minimize phototoxicity. In the case of a multi-view based on four-objective geometry, a large open volume (40 x 20 x 8 mm3) exists between lenses, to observe model organisms (like Zebrafish) under visual and optogenetic stimulation. Such a volume also provides smaller model organisms (C. elegans and Drosophila larvae) with ample space to navigate freely in 3D, while whole brain neural activity is monitored and controlled under various external and internal (optogenetic) stimulations.
TranSIM (Trans?Sheet Illumination Microscope) is an innovative, ultra?fast microscope developed at UCLA. It will be used to quantitatively study whole brain activity in behaving model animals in real time. This research should contribute to our fundamental understanding about the nature and behavior of the brain in the processing of information in health and disease.