In Vivo and in Situ Spectroscopic Imaging by a Handheld Stimulated Raman Scattering Microscope

Spectroscopic stimulated Raman scattering (SRS) microscopy is a label-free technique that generates chemical maps of live cells or tissues. A handheld SRS imaging system using an optical fiber for laser delivery will further enable in situ and in vivo compositional analysis for applications such as medical diagnosis and surgical guidance. In fiber-delivered SRS, the interaction of two ultrashort pulses in the confined mode area creates a significant background that overwhelms the stimulated Raman signal from a sample. Here, we report the first background-free fiber-delivered handheld SRS microscope for in situ chemical imaging. By temporally separating the two ultrafast pulses propagating in the fiber and then overlapping them on a sample through a highly dispersive material, we detected a stimulated Raman signal that is 200 times weaker than the background induced by the fiber. Broad applications of the handheld SRS microscope were demonstrated through in situ ambient-light chemical mapping of pesticide on a spinach leaf, cancerous tissue versus healthy brain tissue in a canine model, and cosmetic distribution on live human skin. A lab-built objective lens further reduced the size of the pen-shaped microscope to about one centimeter in diameter.