Abstract An exciting recent development for high spatial resolution deep tissue imaging is long wavelength three- photon fluorescence microscopy (3PM). Since its first demonstration of imaging subcortical structures in the mouse brain, 3PM has driven rapid progress in deep tissue imaging beyond the depth limit of two-photon fluorescence microscopy (2PM). Long-wavelength 3PM is perhaps the most promising new technology for deep imaging within scattering biological tissues, and has potential impacts in a large number of biomedical fields such as neuroscience, immunology, and cancer biology. On the other hand, there are a number of challenges that must be overcome before 3PM can reach its full potential. Because it is a higher-order nonlinear process, three- photon excitation (3PE) is inherently weaker than two-photon excitation (2PE). The weak signal strength of 3PM is particularly problematic for fast imaging of dynamic cellular process. Furthermore, the laser sources for 3PM are not yet optimized for deep tissue penetration, and the complexity and cost of the excitation source is a major barrier for the applications of 3PM in a typical biomedical research lab. Finally, nearly all 3PM applications today are in the brains. Reaching anatomical frontiers is equally possible in other organs with 3PM, but explicit demonstrations of intravital imaging in novel locations are needed to bring deep imaging capability to other biological systems. The research activity of this proposal will directly address the above challenges for in vivo deep tissue 3PM. We will develop a new generation of 3PM that will improve the performance of existing 3PM by two orders of magnitude and enable multi-color deep tissue imaging with a single excitation wavelength. We will demonstrate the unprecedented imaging capabilities with a low-cost, fiber-based laser system, removing a key barrier for the deployment of 3PM in biology labs. Furthermore, by applying our techniques to a wide variety of biological systems, we will create a valuable knowledge base for the applications of 3PM. Our development of the next generation 3PM parallels the development of 2PM, where the concerted development effort in lasers, microscopes, and biological applications in the 1990s made 2PM ubiquitous in biomedical research labs by the early 2000s. Our vision is to make deep, fast 3PM a routine instrument for a wide variety of biomedical applications just as 2PM does in the shallower regions of biological tissues and organs. The successful completion of this program will enable visualization of dynamic process at the sub-cellular level in intact organs and animal models that are completely beyond the reach of any existing imaging techniques.

Narrative Non-invasive optical imaging is essential for understanding biological functions at the cellular or sub- cellular level. This project brings together an interdisciplinary team to develop the next generation microscopy techniques for imaging deep and fast within intact tissues and organs.