
Three-photon excited fluorescence microscopy enables high-contrast, high-resolution imaging of blood flow, neural structure, and inflammatory response in the mouse spinal cord up to 550 μm in depth, providing a powerful tool to study diverse cellular dynamics in the spinal cord in vivo.


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Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 μm into the mouse spinal cord, in vivo . We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 μm of the murine spinal cord, in vivo .

### Competing Interest Statement

The authors have declared no competing interest.

Three-photon excited fluorescence microscopy enables imaging of blood flow, neural structure and inflammatory response deep into mouse spinal cord in vivo

three-photon-excited-fluorescence-microscopy-enables-comprehensive-imaging-of-spinal-cord-vasculature-neural-structure-and-inflammatory-response-in-living-mice


Three-Photon Excited Fluorescence Microscopy Enables Comprehensive Imaging of Spinal Cord Vasculature, Neural Structure, and Inflammatory Response in Living Mice
