Multi-Scale Light-Sheet Fluorescence Microscopy for Fast Whole Brain Imaging.

Whole-brain imaging has become an increasingly important approach to investigate neural structures, such as somata distribution, dendritic morphology, and axonal projection patterns. Different structures require whole-brain imaging at different resolutions. Thus, it is highly desirable to perform whole-brain imaging at multiple scales. Imaging a complete mammalian brain at synaptic resolution is especially challenging, as it requires continuous imaging from days to weeks because of the large number of voxels to sample, and it is difficult to acquire a constant quality of imaging because of light scattering during in toto imaging. Here, we reveal that light-sheet microscopy has a unique advantage over wide-field microscopy in multi-scale imaging because of its decoupling of illumination and detection. Based on this observation, we have developed a multi-scale light-sheet microscope that combines tiling of light-sheet, automatic zooming, periodic sectioning, and tissue expansion to achieve a constant quality of brain-wide imaging from cellular (3 µm × 3 µm × 8 µm) to sub-micron (0.3 µm × 0.3 µm × 1 µm) spatial resolution rapidly (all within a few hours). We demonstrated the strength of the system by testing it using mouse brains prepared using different clearing approaches. We were able to track electrode tracks as well as axonal projections at sub-micron resolution to trace the full morphology of single medial prefrontal cortex (mPFC) neurons that have remarkable diversity in long-range projections.

Morphological diversity of single neurons in molecularly defined cell types.

Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.