Hyperspectral Coherent Raman Scattering (CRS) Microscopy Based on a Rapidly Tunable and Environmentally Stable Fiber Laser

Abstract

Coherent Raman scattering (CRS) microscopy is an attractive label-free imaging method providing high chemical sensitivity, sub-micron spatial resolution, and video-rate imaging speed. When extended to hyperspectral CRS (HS-CRS), it is capable of distinguishing molecules even in complex heterogeneous systems by probing multiple Raman resonances. Solid-state lasers, associated with their high cost, large footprint, and slow tuning speed, currently limit the widespread application of HS-CRS. Fiber lasers with comparable properties usually suffer from poor noise performance or instabilities during wavelength tuning. A tunable two-color fiber laser is demonstrated that can be electronically tuned at rates of up to 1 kHz. The use of polarization-maintaining fibers and an all-optical synchronization mechanism guarantees its mode-locking stability. The superior noise performance of this system is evaluated by the side-by-side comparison with data obtained by a commercial optical parametric oscillator (OPO)-based laser. Large-area multimodal imaging of liver tissue sections surgically resected from a patient suffering from a non-neoplastic liver parenchyma with micronodular cirrhosis in combination with a multilocular hepatocellular carcinoma is demonstrated. By exploiting the fast-hyperspectral scanning capability of the laser and multivariate statistical analysis, virtual staining of tissue sections and a comparison of the diagnostic properties of HS-CRS to the classical histopathological approach are achieved.