Real-Time Microscale Temperature Imaging by Stimulated Raman Scattering

Microscale thermometry of aqueous solutions is essential to understand the dynamics of local heat generation and dissipation in chemical and biological systems. A wide variety of fluorescent probes have been developed to map temperature changes with submicrometer resolution, but they often suffer from the uncertainty associated with microenvironment-dependent fluorescent properties. In this work, we develop a label-free ratiometric stimulated Raman scattering (SRS) microscopy technique to quantify microscale temperature by monitoring the O-H Raman stretching modes of water. By tracking the ratio changes of the hydrogen-bonding O-H band and the isosbestic band, we can directly quantify the temperature of water-based environments in real time without exogenous contrast agents. We demonstrate real-time measurement of localized intracellular and extracellular temperature changes due to laser absorption. This high-speed nonlinear optical imaging technique has the potential for in situ microscale imaging of thermogenesis in both chemical and biological systems.