<i>In Vivo</i> and <i>in Situ</i> Spectroscopic Imaging by a Handheld Stimulated Raman Scattering Microscope Chien-Sheng Liao Pu Wang Chih Yu Huang Peng Lin Gregory Eakins R. Timothy Bentley Rongguang Liang Ji-Xin Cheng 10.1021/acsphotonics.7b01214.s002 https://acs.figshare.com/articles/media/_i_In_Vivo_i_and_i_in_Situ_i_Spectroscopic_Imaging_by_a_Handheld_Stimulated_Raman_Scattering_Microscope/5783076 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. 2017-12-27 00:00:00 fiber-delivered ultrafast pulses propagating ambient-light chemical mapping sample SRS microscope tissue application Situ Spectroscopic Imaging lab-built objective lens Raman signal background Handheld Stimulated Raman Scattering Microscope Spectroscopic SRS imaging system