Shot-Noise-Limited Two-Color Stimulated Raman Scattering Microscopy with a Balanced Detection Scheme

Stimulated Raman scattering (SRS) microscopy has been considered a useful technique for investigating chemical components by selectively targeting the vibration mode of chemical structures. Its practical application to the observation of molecular structures and dynamics in complicated biological environments requires broad spectral coverage with both high resolution and a high signal-to-noise ratio. Here, we demonstrate a two-color SRS microscopy employing a balanced detection scheme and a spectral focusing method. Two different SRS signals are generated with pump and Stokes laser pulse pairs in perpendicular polarization, where each of them acts as an intensity reference for the other, significantly reducing the background noise level close to the shot-noise limit even with a fiber-based femtosecond laser system. The high spectral resolution comparable to that of spontaneous Raman scattering spectroscopy is achieved with the spectral focusing method. The two-color SRS images are obtained for a mixture of polymer beads and for the distributions of lipids and proteins in U2OS cells.