High-speed super-multiplex imaging of brain tissue

Cells within the brain are highly organized and coordinate complex processes with each other. The ability to simultaneously visualize the organization and interactions of cells and molecules within brain tissue remains an important issue to understand the brain comprehensively. Stimulated Raman scattering (SRS) and fluorescence, two powerful imaging modalities, can provide complementary molecular contrasts within cells and tissue samples. Here, we present a high-speed super-multiplex imaging platform that combines SRS microscopy with confocal fluorescence microscopy to perform rapid 7-color brain imaging. We show simultaneous imaging of cellular components within the brain such as astrocytes, axons, and blood vessels while also showing organelles such as the nucleus and actin cytoskeleton. Also, we demonstrate the ability to take depth-resolved images that elucidate the three-dimensional organization of diverse components within brain tissue. This platform can be adapted to explore various processes within brain tissue that can reveal critical information about the brain and how it is affected by diseases, which leads toward a deeper understanding of disease progression and potentially the development of therapeutic options for brain diseases.