Advantages of three-photon live imaging for deep tissue analysis.

Introduction: Three-photon microscopy is an emerging tool for deep tissue imaging with superior spatial resolution. It enables imaging of portions of tissue beyond the typical depth limit of two-photon microscopy.

Areas covered: In this review, we give an overview of widely used deep tissue imaging modalities. We highlight the advantages of three-photon microscopy and review its current applications in biomedical research as well as its potential pre-clinical and clinical applications. Finally, we assess the challenges in the widespread use of three-photon live imaging in biomedical research and in clinical translation.

Expert opinion: Three-photon microscopy holds great promise as a biomedical research tool. It can uncover insights into the structure and function of tissues beyond what is accessible with two-photon microscopy. It also has promise to be a powerful clinical tool. Future clinical applications include optical biopsies with near instantaneous results and guided surgical resections. A multimodal strategy that combines three-photon technology with a large field of view, low resolution technique may be used to overcome the limited field of view of three-photon microscopy for macroscopic tissue assessment.