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.