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

Area of Science:

• Neuroscience
• Biomedical Engineering
• Optical Imaging

Background:

• Miniature multiphoton microscopy offers advanced neuronal imaging in freely behaving animals but faces limitations in field of view and depth of field, hindering precise region identification in 3D space.
• Current systems' shallow depth of field and limited field of view (FOV) pose challenges for researchers in accurately locating regions of interest across large cranial windows, impacting usability.

Purpose of the Study:

• To develop an advanced multimodal imaging platform with improved guidance and a standardized workflow for efficient imaging of freely behaving animals.
• The goal was to overcome the limitations of existing miniature multiphoton microscopy systems by enhancing ease of use and precision in identifying target regions.

Main Methods:

• Introduced a wide-field fluorescence navigation system (WF-Nav) with a 90-mm working distance, 4-mm FOV, and single-cell resolution for rapid region localization.
• Integrated WF-Nav with miniature two- and three-photon microscopes to create a versatile multimodal platform, enabling seamless transitions between imaging modalities.
• Developed a streamlined, user-friendly workflow specifically designed for imaging freely behaving mice.

Main Results:

• Validated the platform through large-FOV (4 mm), dual-color (920 and 1030 nm), and deep-brain (up to 1 mm) neuronal imaging in awake and freely moving mice.
• Demonstrated high efficiency and success rates, with the entire experimental procedure completed in approximately 20 minutes and a 100% success rate (n=15).

Conclusions:

• Developed a comprehensive imaging platform integrating single-photon wide-field navigation with miniature two- and three-photon microscopy.
• Established a streamlined workflow for imaging freely behaving animals, significantly enhancing the platform's applicability and operational efficiency.
• The multimodal approach leverages the strengths of each imaging modality, offering a powerful tool for neuroscience research.