Volumetric chemical imaging by clearing-enhanced stimulated Raman scattering microscopy

Significance Cells form structures and perform functions through intricate 3D tissue organizations. However, due to tissue scattering, coherent Raman microscopy—a powerful method complementary to fluorescence imaging—suffers from limited imaging depth in tissues. Here, we develop a volumetric chemical imaging method with greater than 10-fold depth increase. We formulate a Raman-tailored tissue-clearing recipe and combine it with advanced Raman microscopies. Equipped with the toolbox of volumetric chemical imaging and analyses, we elucidate complex 3D structures, chemical compositions, and metabolic dynamics in diverse tissues including lipid synthesis throughout tumor spheroids, 3D networks of axons, vasculatures, and cell bodies in brain regions, as well as heterogeneous tumor structures and tumor metabolism. Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.

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