Abstract The goal of this project is to develop a novel bifocal catadioptric objective that will allow large-scale recording of neural circuits in vivo. The objective will enable faster volumetric imaging of large brain regions by simultaneous two-photon (2P) imaging of shallow layers and three-photon (3P) imaging of deep layers of brain tissues with improved collection efficiency. Although three-photon microscopy (3PM) allows imaging at depths inaccessible by two photon microscopy (2PM), 2P excitation generates larger fluorescence signal with lower excitation pulse energy when imaging at shallow tissue layers. Therefore, for fast imaging across a large depth, the optimum approach is to use 2PM for the superficial layers and 3PM for the deeper regions simultaneously. Implementation of this approach inevitably requires the objective lens to generate two focal planes that are separated by a large axial distance while still maintaining high spatial resolution and large field of view. Simultaneous 2PM and 3PM will not only allow for utilization of the advantages of both modalities but also for faster volumetric imaging of large brain columns. We will develop a bifocal catadioptric (i.e., both refractive and reflective) lens based on the idea of separation the optical paths of the excitation light with different wavelengths. The lens will feature two focal planes separated axially by ~ 600 µm for the 2P (< 1100 nm) and 3P (>1200 nm) excitation wavelengths. The design approach also separates the excitation path and the collection path and allows independent optimization for efficient collection of the emitted fluorescence. The bifocal objective will collect fluorescence back through non-imaging pathways, which enables the proposed catadioptric objective to have a large collection numerical aperture and a large collection field of view. The collection efficiency is approximately 5x higher than the commercially available objective lenses when imaging deep (>1 mm) into the mouse brain. Improving the signal collection efficiency will immediately increase the frame rate without increasing the excitation power, enabling high-resolution, high-speed imaging at these depths. We will design, fabricate and validate the novel objective lens and will combine it with focus-tunable lenses to enable faster volumetric imaging of mouse brains. The successful completion of this program will immediately enable simultaneous 2P and 3P imaging across a large range of depth (~ 1.2 mm), such as recording population of neurons across different layers of mouse brains. The technology developed within this program will have potential impacts in a large number of biomedical fields such as neuroscience, immunology, and cancer biology.

Narrative The proposed program will lead to a novel bifocal objective that will allow fast imaging of neuronal activity in large brain volumes by simultaneous two-photon imaging of shallow and three-photon imaging of deep tissue layers with improved collection efficiency. The technology created in this program will seamlessly integrate two- and three-photon imaging modalities for recording brain activities across a large range of depth simultaneously.