Abstract
Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one‐shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. 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 demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. 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. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.
Highlights
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
In chromatic multiphoton serial (ChroMS) microscopy, the sample is crosslinked with an embedding agarose block, and large-scale imaging is achieved by automatically alternating simultaneous multimodal acquisitions, stage-based lateral mosaicking, and tissue sectioning with a vibrating-blade microtome
We present here a multicolor/multi-contrast tissue-scale microscopy method which bypasses the spectral limitation common to current large volume imaging techniques, and in particular expands the field of applications of serial two-photon microscopy[23,44]
Summary
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. 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[1,2,3,4,5,6,7,8,9], cell lineage[10,11,12,13,14,15], cycling state[16,17], subtype identity[18], genotype[19,20], or signaling pathway activation[21] 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. Achieving tissue-scale multicolor microscopic imaging requires addressing chromatic aberrations and channel registration over large volumes 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. We illustrate its potential for high informationcontent 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
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