Multicontrast organ-scale imaging with ChroMS microscopy We illustrate its potential for high information-content three-dimensional (3D) imaging by demonstrating (i) analysis of astroglial cell morphology and contacts over several mm 3 of cerebral cortex, (ii) color-assisted tracing of tens of pyramidal neurons in a densely labeled, mm-thick cortical sample, and (iii) brain-wide color-based multiplexed mapping of axonal projection trajectories and interdigitation. In addition to being perfectly suited for applications based on combinatorial fluorescence labeling, ChroMS microscopy also enables organ-wide imaging of label-free nonlinear signals such as third harmonic generation (THG) and coherent anti-Stokes Raman scattering (CARS). We demonstrate its performance for tridimensional imaging of mouse brains labeled by transgenic, electroporation-based or viral multicolor approaches. We show that ChroMS microscopy delivers multicolor imaging over >mm 3 volumes with constant micron-scale resolution and submicron channel coregistration, which sets new quality standards for large-scale multicolor microscopy. Our approach, termed chromatic multiphoton serial (ChroMS) microscopy, relies on the integration of trichromatic two-photon excitation by wavelength mixing (WM) 30 with automated serial tissue sectioning. Here, we report on a method for volume multicolor and multi-contrast microscopy with submicrometer registration of the image channels. These obstacles made it so far very difficult to probe cell interactions or more generally to image multiplexed or combinatorial fluorescent signals with micrometer-scale precision in samples exceeding a few hundreds of microns in depth. In addition, achieving tissue-scale multicolor microscopic imaging requires addressing chromatic aberrations and channel registration over large volumes. This limitation is due to the general difficulty of exciting a manifold of fluorescent proteins. However, microtome-assisted microscopy methods developed so far are limited to single- or dual-color imaging 23, 24, 26, 29. One particularly active field of application is neuroscience, where block-face fluorescence imaging has opened the way to brain-wide mesoscale connectomics 23, 26 and single neuron reconstruction efforts 26– 29. This scheme has emerged as an effective means for generating data encompassing mm 3-to-cm 3 volumes of tissue at subcellular-resolution with either discrete or continuous sampling. In recent years, serial block-face imaging, which relies on the automated, iterative alternation of imaging and microtome-based sectioning of a block of tissue, has been successfully transposed from electron microscopy to light microscopy 22– 25. Scaling up such approaches at the whole organ or tissue level would be a major step forward but is hindered by the lack of suitable large-volume multicolor microscopy methods. Strategies combining multiple distinct fluorescent labels are increasingly used to study spatial relationships among cells and molecules and to encode parameters such as neuronal connectivity 1– 9, cell lineage 10– 15, cycling state 16, 17, subtype identity 18, genotype 19, 20, or signaling pathway activation 21. Multicolor fluorescence microscopy has become a key enabling technology in biology by providing the means to spectrally resolve cells, organelles, or molecules within tissues and to analyze their interactions. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one‐shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities.
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