Chemical imaging of tissue <i>in vivo</i> with video-rate coherent anti-Stokes Raman scattering microscopy

TLDR

Imaging living organisms with molecular selectivity usually requires exogenous labels, but many biological and medical applications would benefit from a noninvasive technique; in vivo microscopy using vibrational spectroscopic contrast provides a unique way to visualize tissue architecture with molecular specificity. The authors developed a sensitive, video‑rate coherent anti‑Stokes Raman scattering (CARS) technique that uses backscattered forward‑propagating CARS radiation to generate a strong epi‑CARS signal, enabling in vivo vibrational imaging. The strong epi‑CARS signal permits real‑time monitoring of dynamic processes, and by tuning to the CH₂ stretching band the authors achieved high‑contrast, subcellular‑resolution imaging and spectroscopy of lipid‑rich structures in live mouse skin.

Abstract

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH 2 stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.