Simultaneous Dual-Band Hyperspectral Stimulated Raman Scattering Microscopy with Femtosecond Optical Parametric Oscillators.

Stimulated Raman scattering (SRS) microscopy is a label-free quantitative optical technique for imaging molecular distributions in cells and tissues by probing their intrinsic vibrational frequencies. Despite its usefulness, existing SRS imaging techniques have limited spectral coverage due to either a wavelength tuning constraint or narrow spectral bandwidth. High-wavenumber SRS imaging is commonly used to map lipid and protein distribution in biological cells and visualize cell morphology. However, to detect small molecules or Raman tags, imaging in the fingerprint region or "silent" region, respectively, is often required. For many applications, it is desirable to collect SRS images in two Raman spectral regions simultaneously for visualizing the distribution of specific molecules in cellular compartments or providing accurate ratiometric analysis. In this work, we present an SRS microscopy system using three beams generated by a femtosecond oscillator to acquire hyperspectral SRS image stacks in two arbitrary vibrational frequency bands, between 650-3280 cm, simultaneously. We demonstrate potential biomedical applications of the system in investigating fatty acid metabolism, cellular drug uptake and accumulation, and lipid unsaturation level in tissues. We also show that the dual-band hyperspectral SRS imaging system can be adapted for the broadband fingerprint region hyperspectral imaging (1100-1800 cm) by simply adding a modulator.