In Vivo Simultaneous Nonlinear Absorption Raman and Fluorescence (SNARF) Imaging of Mouse Brain Cortical Structures

A bstract Two-photon excited fluorescence (TPEF) microscopy is a widely used optical imaging technique that has revolutionized neurophotonics through a diverse palette of dyes, specialized transgenic models, easy implementation, and straightforward data analysis. However, in vivo TPEF imaging is often limited in the number of contrasts available to distinguish different cells, structures, or functions. We propose using two label-free multiphoton microscopy techniques – stimulated Raman scattering (SRS) microscopy and transient absorption microscopy (TAM) – as complementary and orthogonal imaging modalities to TPEF for in vivo brain imaging. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope and image several cortical structures up to 250-300 μm below the pial surface, the highest reported in vivo imaging depth for SRS or TAM. We further demonstrate the capabilities of our SNARF microscope through the quantification of age-dependent myelination, hemodynamics, vessel structure, cell density, and cell identity in vivo. Using machine learning, we report the use of label-free SRS and TAM features to predict capillary-lining cell identities with 90% accuracy. The SNARF microscope and methodology outlined herein provide a powerful platform to study several research topics, including neurovascular coupling, blood-brain barrier, neuronal and axonal degeneration in aging, and neurodegenerative diseases.