Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy

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. We thank Minh-Son Phan, Samuel Tozer, and Z. Josh Huang for scientific discussions and comments on the manuscript. We thank TissueVision for providing technical advice for the implementation and customization of the block-face system. We thank Bastien Binet from Polytechnique Polymedia center for his generous help in the creation of 3D graphics. We thank IdV core imaging, animal experimentation and histology facilities for technical assistance. This work was supported by fellowships from Universite Paris-Saclay (Initiatives Doctorales Interdisciplinaires) to L.A., Ecole des Neurosciences de Paris to K.M., Region Ile-de-France and Fondation ARC pour la Recherche sur le Cancer to S.C. by Agence Nationale de la Recherche under contracts ANR-11-EQPX-0029 (Equipex Morphoscope2), ANR-10-INBS-04 (France BioImaging), and ANR-10-LABX-65 (LabEx LifeSenses), by Fondation pour la Recherche Medicale (grant DBI20141231328), by Target ALS and by the European Research Council (ERC-CoG 649117).