Nonlinear hyperspectral imaging of collagen by vibrational sum-frequency generation microscopy (Invited Paper)

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 µm. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~ 1 µm level, and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. In this study, we demonstrate the capability of VSFG microscopy to discern chemically specific domain details of collagen in both mouse lung tumor and control tissues. We introduce two methods for identifying the tumor domain using chemical-specific VSFG imaging. These findings underscore the potency of VSFG microscopy as a transformative tool in the realm of bioimaging for medical research in both revealing fundamental structural of collagens and as a diagnostic tool in clinical setting.