iSPIM, SCAPE/OPM and line scan confocal microscopy are partially effective in alleviating this limitation, however at the cost of reduction in usable working distance ( magenta arrowheads) and image quality (e.g., SCAPE collects low-quality signal from non-native focal planes, and line scan confocal results in lower axial resolution and high photo-bleaching.). a Light sheet microscopy ( LSM) employs orthogonally illumination-detection optics, which limits the lateral dimensions of imaging volume. Light sheet theta microscopy ( LSTM) for high-resolution quantitative imaging of large intact samples. Similar attempts of LSM imaging of clarified rat brains resulted in much poorer image quality in large parts of the brain. For example, we previously reported an optimized implementation of LSM, called CLARITY optimized light sheet microscopy (COLM), which allowed high-resolution imaging of entire intact mouse brains in a few hours imaging time, although with progressively reduced image quality towards the middle of the samples due to the scattering of illumination light sheets. However, these advantages of orthogonal illumination-detection geometry also require unhindered optical access from the sides of the samples, thus limiting the lateral dimensions (along the illumination light sheet) of the imaging volumes (Fig. On the other hand, light sheet microscopy (LSM)-based approaches, with their orthogonal single-plane illumination and simultaneous whole-plane detection, are proving to be highly effective due to minimal photo-bleaching and high imaging speeds (2–3 orders of magnitude more than confocal). However, these approaches still entail highly redundant illumination of out-of-focus parts of the samples and also reduced axial resolution and imaging depth, thus limiting their utility for high-resolution imaging of large cleared samples such as whole mouse brains (Fig. ![]() Variants of confocal microscopy, including line scanning confocal microscopy (LSCM), can provide much higher imaging speeds due to parallel imaging of multiple points. However, taking full advantage of these techniques requires rapid high-resolution three-dimensional (3D) imaging of very large volumes.Ĭonventional point-scanning approaches, such as confocal and two-photon microscopy, provide high imaging quality, but their slow imaging speeds and high photo-bleaching rates render them less effective for imaging of large volumes. ![]() These approaches have the potential to accelerate discoveries across multiple domains of life sciences, including an understanding of the mammalian brain architecture, reconstructing tumor microenvironments, and in situ transcriptomics. Furthermore, the development of physical tissue expansion approaches (expansion microscopy, ExM ) is enabling higher (super-resolution) effective imaging resolutions, although at the cost of ever-increasing sample sizes (up to 20-fold expansion demonstrated ). Most of these approaches employ a cocktail of chemicals for cellular membrane lipid dissolution and/or refractive index smoothening to render the tissue transparent. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.Īdvances in tissue clearing methods are enabling unhindered optical access to the structure and function of large intact biological systems such as mouse brain and tumor biopsies. ![]() We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems.
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