Second harmonic generation imaging is a powerful tool for visualizing molecular structures in living organisms without the need for exogenous dyes. However, SHG signal lacks molecular specificity in identifying the source. This study aimed at molecular identification of SHG sources in the mouse brain using a multimodal imaging technique combining SHG and multiplex coherent anti Stokes Raman scattering (CARS) spectroscopy. We performed multimodal imaging in two different regions, the surface and dentate gyrus of the brain tissue. For the brain surface, the SHG signal was recognized through CARS spectrum analysis, indicating its origin in collagen. In the dentate gyrus, CARS images did not unveil corresponding molecular origins; however, morphologically, the SHG signal likely originated from Rootletin within neurons. Overall, Multimodal imaging approach to molecular identification of SHG has the potential to contribute to a comprehensive understanding of the molecular and structural features of the mouse brain. These findings advance label-free imaging techniques and have implications for brain tissue analysis and functional mapping research.
Mapping the distribution of chemical molecules throughout a brain is helpful for neuroscience research. We have applied an ultra-broadband multiplex CARS spectroscopic imaging system to construct a whole-brain label-free molecular map in macro and micro scales. We could precisely visualize lipids distributed to white matter, rich in neuronal fibers. Our microscale measurements figured out that cells within the hippocampus and cerebral cortex could be divided into lipid-rich and water-rich cells. Moreover, we applied multivariate curve decomposition analysis for our spectrum and recapitulated the results. Our imaging and analysis techniques will lead to the molecular brain atlas with single-cell resolution.
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