<i>In Vivo</i> and <i>in Situ</i> 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 <i>in situ</i> and <i>in vivo</i> 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 <i>in situ</i> 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 <i>in situ</i> 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.