Spectroscopic second and third harmonic generation microscopy using a femtosecond laser source in the third near-infrared (NIR-III) optical window.

In this study, second harmonic generation (SHG) and third harmonic generation (THG) spectroscopic imaging were performed on biological samples using a femtosecond laser source in the third near-infrared (NIR) optical window (NIR-III). Using a visible-NIR spectrometer, the SHG and THG signals were simultaneously detected and were extracted using spectral analysis. Visualization of biological samples such as cultured cells (HEK293 T), mouse brain slices, and the nematode Caenorhabditis elegans was performed in a label-free manner. In particular, in an SHG image of an entire coronal brain section (8 × 6 mm2), we observed mesh-like and filamentous structures in the arachnoid mater and wall of the cerebral ventricle, probably corresponding to the collagen fibers, cilia, and rootlet. Moreover, the THG images clearly depicted the densely packed axons in the white matter and cell nuclei at the cortex of the mouse brain slice sample and lipid-rich granules such as lipid droplets inside the nematode. The observations and conclusions drawn from this technique confirm that it can be utilized for various biological applications, including in vivo label-free imaging of living animals.