Tissue imaging depth limit of stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is a promising technique for studying tissue structure, physiology, and function. Similar to other nonlinear optical imaging techniques, SRS is severely limited in imaging depth due to the turbidity and heterogeneity of tissue, regardless of whether imaging in the transmissive or epi mode. While this challenge is well known, important imaging parameters (namely maximum imaging depth and imaging signal to noise ratio) have rarely been reported in the literature. It is also important to compare epi mode and transmissive mode imaging to determine the best geometry for many tissue imaging applications. In this manuscript we report the achievable signal sizes and imaging depths using a simultaneous epi/transmissive imaging approach in four different murine tissues; brain, lung, kidney, and liver. For all four cases we report maximum signal sizes, scattering lengths, and achievable imaging depths as a function of tissue type and sample thickness. We report that for murine brain samples thinner than 2 mm transmissive imaging provides better results, while samples 2 mm and thicker are best imaged with epi imaging. We also demonstrate the use of a CNN-based denoising algorithm to yield a 40 µm (24%) increase in achievable imaging depth.