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 similar to 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.