ClearScope: a fully integrated light sheet theta microscope for sub-cellular resolution imaging without lateral size constraints

Three-dimensional (3D) ex vivo imaging of cleared intact brains of animal models and large human and non-human primate postmortem brain specimens is important for understanding the physiological neural network connectivity patterns and the pathological alterations underlying neuropsychiatric and neurological disorders. Light-sheet microscopy has emerged as a highly effective imaging modality for rapid high-resolution imaging of large cleared samples. However, the orthogonal arrangements of illumination and detection optics in light sheet microscopy limits the size of specimen that can be imaged. Recently developed light sheet theta microscopy (LSTM) technology addressed this by utilizing a unique arrangement of two illumination light paths oblique to the detection light path, while allowing perpendicular arrangement of the detection light path relative to the specimen surface. Here, we report development of a next-generation, fully integrated, and user-friendly LSTM system for rapid sub-cellular resolution imaging uniformly throughout a large specimen without constraining the lateral (XY) size. In addition, we provide a seamlessly integrated workflow for image acquisition, data storage, pre- and post-processing, enhancement, and quantitative analysis. We demonstrate the system performance by high-resolution 3D imaging of intact mouse brains and human brain samples, and complete data analysis including digital neuron tracing, vessel reconstruction and design-based stereological analysis in 3D. This technically enhanced and user-friendly LSTM implementation will enable rapid quantitative mapping of molecular and cellular features of interests in diverse types of very large samples.