This study presents a novel method for in vivo chemical imaging of tissue using video-rate coherent anti-Stokes Raman scattering (CARS) microscopy. CARS is a nonlinear optical technique that provides molecular specificity by exploiting vibrational transitions in molecules. The researchers combined CARS with video-rate microscopy to enable real-time imaging of live tissues with high sensitivity and resolution. The technique relies on the backscattering of intense forward-propagating CARS radiation in tissue, which generates a strong epi-CARS signal. This signal allows for the visualization of subcellular structures in live tissues with unprecedented contrast. The method was applied to imaging lipid-rich structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes. The CARS signal was tuned to the CH₂ stretching vibrational band, enabling the detection of lipid-rich regions with high specificity. The technique was also used to track the diffusion of chemical compounds, such as mineral oil, through the skin in real time. The study demonstrates that CARS microscopy can provide chemical selectivity and high-resolution imaging of tissues in vivo. It is particularly effective for visualizing lipid distributions, as CARS is sensitive to the vibrational modes of CH₂ groups. The technique was also combined with two-photon fluorescence microscopy to provide additional information about tissue structures. The researchers also showed that CARS can distinguish between different tissue components based on their chemical composition. For example, sebaceous glands and adipocytes were distinguished by their distinct CARS signals at different Raman shifts. The study highlights the potential of CARS for biomedical applications, including real-time imaging of tissue pathology and molecular imaging of intravascular plaques. The method was validated using both in vivo and ex vivo experiments, with the results demonstrating the high sensitivity and resolution of CARS imaging. The study also addresses the challenge of imaging deep tissues, showing that the penetration depth is limited by the working distance of the microscope objective. However, the technique has the potential to be improved further with advancements in detection sensitivity and laser technology. The results suggest that CARS microscopy could be used for real-time imaging of proteins and DNA in the future.This study presents a novel method for in vivo chemical imaging of tissue using video-rate coherent anti-Stokes Raman scattering (CARS) microscopy. CARS is a nonlinear optical technique that provides molecular specificity by exploiting vibrational transitions in molecules. The researchers combined CARS with video-rate microscopy to enable real-time imaging of live tissues with high sensitivity and resolution. The technique relies on the backscattering of intense forward-propagating CARS radiation in tissue, which generates a strong epi-CARS signal. This signal allows for the visualization of subcellular structures in live tissues with unprecedented contrast. The method was applied to imaging lipid-rich structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes. The CARS signal was tuned to the CH₂ stretching vibrational band, enabling the detection of lipid-rich regions with high specificity. The technique was also used to track the diffusion of chemical compounds, such as mineral oil, through the skin in real time. The study demonstrates that CARS microscopy can provide chemical selectivity and high-resolution imaging of tissues in vivo. It is particularly effective for visualizing lipid distributions, as CARS is sensitive to the vibrational modes of CH₂ groups. The technique was also combined with two-photon fluorescence microscopy to provide additional information about tissue structures. The researchers also showed that CARS can distinguish between different tissue components based on their chemical composition. For example, sebaceous glands and adipocytes were distinguished by their distinct CARS signals at different Raman shifts. The study highlights the potential of CARS for biomedical applications, including real-time imaging of tissue pathology and molecular imaging of intravascular plaques. The method was validated using both in vivo and ex vivo experiments, with the results demonstrating the high sensitivity and resolution of CARS imaging. The study also addresses the challenge of imaging deep tissues, showing that the penetration depth is limited by the working distance of the microscope objective. However, the technique has the potential to be improved further with advancements in detection sensitivity and laser technology. The results suggest that CARS microscopy could be used for real-time imaging of proteins and DNA in the future.