Wide-field fluorescence navigation system for efficient miniature multiphoton imaging in freely behaving animals.

Significance: Miniature multiphoton microscopy has revolutionized neuronal imaging in freely behaving animals. However, its shallow depth of field-a result of high axial resolution-combined with a limited field of view (FOV), makes it challenging for researchers to identify regions of interest in three-dimensional space across multimillimeter cranial windows, thereby reducing the system's ease of use.

Aim: We aimed to develop a multimodal imaging platform with enhanced guidance and a standardized workflow tailored for efficient imaging of freely behaving animals.

Approach: We present a wide-field fluorescence navigation system (WF-Nav) featuring a 90-mm working distance, a 4-mm FOV, and single-cell resolution, enabling rapid and precise localization of designated regions. By seamlessly integrating this navigation system with our prior miniature multiphoton microscopes, we established a multimodal platform that supports versatile imaging modalities and seamless transitions to two- or three-photon imaging. Building on this integration, we developed a streamlined workflow for efficient, user-friendly imaging in freely behaving mice.

Results: We validated the system through large-FOV imaging (4 mm), dual-color imaging (920 and 1030 nm), and deep-brain neuronal imaging (up to 1 mm) in either awake mice or freely moving mice. The entire experimental procedure was completed in $\sim 20\min$ , achieving a 100% success rate ( $n=15$ ).

Conclusions: We have developed a comprehensive imaging platform that integrates a single-photon wide-field navigation system with miniature two-photon and three-photon microscopy, leveraging the strengths of each modality. Building on this platform, we established a streamlined workflow tailored for imaging freely behaving animals, markedly expanding its applicability and improving efficiency.