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Wu Runlong's research group develops a wide field fluorescence navigation system to empower efficient multiphoton imaging of freely moving animals
Date: 2025-07-15Read: 4
background
Fluorescence imaging technology, including single photon (1P), two-photon (2P), and three photon (3P) microscopy techniques, has greatly promoted the development of neuroscience. Especially with the emergence of miniaturized fluorescence microscopes, a new paradigm for observing the structure and activity of neurons in freely moving animals has been opened up. Thanks to the high efficiency of single photon excitation, miniaturized single photon microscopes (m1PM) can use LED light sources to achieve illumination in the millimeter range and corresponding imaging field of view (FOV). However, due to the lack of optical sectioning capability, the resolution of m1PM's in vivo imaging is limited, making it impossible to observe fine structures such as dendritic spines. Two photon imaging technology has inherent optical sectioning and deep tissue penetration capabilities, among which miniaturized two-photon microscopy (m2PM) has achieved imaging of neuronal synapses in freely moving mouse brains. The further development of this technology expands the imaging field of view to 1 × 0.8mm2, achieving synchronous tri color imaging and reaching an imaging depth of 854 µ m. In addition, the miniaturized three photon microscope (m3PM) utilizes the three photon effect to achieve imaging depths exceeding 1mm.
Miniature multiphoton microscopes play an increasingly important role in imaging freely moving animals, but their practical applications still face many challenges. The excellent optical slicing ability of multiphoton microscopes results in shallow depth of field (3 to 20 µ m, determined by axial resolution), with imaging fields typically within 1mm2. In the absence of guidance, it is still challenging to quickly locate the region of interest (ROI) of a target in three-dimensional space spanning several millimeters of cranial windows or gradient refractive index lenses (GRIN lenses). Especially for inexperienced researchers, confirming ROI and installing miniaturized microscopes on mouse heads may take tens of minutes or even hours, which greatly limits the further application of this technology in free movement animal research.
To address this issue, the development team combined a wide field (WF) objective with a miniaturized objective (FHIRM-TPM) of the m2PM head mounted device in early work to create a wide field fluorescence imaging device for positioning. However, due to the limited field of view of early miniaturized objectives (about 150 µ m), it was still difficult to quickly locate the region of interest within a few millimeters wide cranial window. In addition, when switching to m2PM imaging, due to the limited working distance of the wide field objective lens (about 37.5mm), significant bending of the fiber is required, making the operation more challenging. Recently, Zong et al. introduced a rotary table to fix the m2PM headset, achieving better alignment with specific brain regions through angle adjustment. Despite these advances, there are still challenges in rapidly locating regions of interest in three-dimensional brain tissue, which presents a steep learning curve for new researchers. In addition, the current working distance of m2/3PM is less than 2mm. When using the z-axis platform to position the focal plane, the sample or objective lens is easily damaged, especially for inexperienced users. Overall, there is an urgent need to develop a multimodal imaging platform with enhanced guidance capabilities and establish standardized workflows to achieve efficient imaging of freely moving animals.
System Design of Multimodal Fluorescence Imaging Platform
To address this issue, the development team designed a multimodal fluorescence imaging platform that integrates a wide field fluorescence navigation system (WF Nav) with miniaturized two-photon microscopy imaging and miniaturized three photon microscopy imaging. To fully leverage the advantages of single photon excitation, the WF Nav system is equipped with a large field of view and single-cell resolution function, which can quickly identify designated areas of interest. Once the target area is located, the system can seamlessly switch to two-photon or three photon imaging, further achieving high signal-to-noise ratio or deep neural imaging. Subsequently, fix the m2PM or m3PM probe on the mouse head and release the mouse for free movement imaging.
Figure 1. Design of Multimodal Fluorescence Imaging System
Design of Wide Field Fluorescence Navigation System
The WF Nav system consists of a multi-color wide field microscope and a multifunctional adapter. The system uses three LEDs as excitation light sources (405nm, 473nm, 561nm) and is equipped with a high numerical aperture non spherical condenser lens to collimate the LED beam. Each excitation light path is equipped with a corresponding filter group. To achieve large field of view navigation and provide sufficient space for seamless switching of multiphoton mirror groups, the development team has designed a dry objective lens with long working distance and low magnification. The objective lens (3.3x) adopts a reverse telephoto design: the front group is composed of two laminated double lenses providing negative optical power, and the rear group is composed of a single spherical lens. This structure shifts the main plane backwards to correct chromatic aberration, achieving a working distance of ultra long effective focal length while maintaining the numerical aperture, ultimately achieving a breakthrough target of 90mm working distance, and achieving a maximum imaging field of view of Φ 5.64mm.
Figure 2. Design of Wide Field Fluorescence Microscope
To achieve seamless switching between wide field mode and multiphoton imaging mode, the team designed a multifunctional adapter. This adapter is connected to a wide field objective lens and adjusts the focal length on a Z-axis electric platform. Through standardized m2/3PM probe design, the probe can be securely mounted on the same bracket, ensuring in focus imaging when switching between multiphoton and wide field modes.
Figure 3. Multi functional adapter and autofocus function
This 90mm working distance objective provides ample space for operating head components, optical fibers, or installing detachable electric zoom lens modules. The head piece bracket is installed on a lightweight linear ball slide rail with minimal sliding resistance (about 0.1N) and a telescopic distance of 12mm, which can effectively avoid the risk of collision between the objective lens and the sample. By replacing the header bracket, the system can be compatible with different headers, such as mini2P and m3PM. In addition, thanks to the size and retractable design of miniaturized two-photon microscopes, compared to conventional desktop objectives, this system provides a larger observation angle in most directions, resulting in a wider sample observation range. This design significantly improves the coarse localization efficiency of the sample.
Figure 4. Comparison of observation angles between different objective lenses and miniaturized two-photon microscopes
Next, the developers evaluated the optical performance and practical applications of the wide field fluorescence microscope. Imaging the brain slices of Thy1 YFPH mice can clearly identify neurons and axons. In vivo calcium ion imaging was performed at a wavelength of 470nm, and morphological imaging was performed at a wavelength of 561nm, successfully capturing 3804 GCaMP6f labeled neurons and 4650 mCherry expressing mouse astrocytes. These results demonstrate that the wide field microscope has multi-color, single-cell resolution, and high-throughput imaging capabilities, and provides excellent performance for navigating target areas of interest.
Video 5. Miniature Wide Field Microscope for High Throughput Imaging
Establishment of standardized micro multiphoton microscopy imaging process
In order to make the imaging of m2PM in freely moving mice smoother, developers have established a standardized imaging process to improve the efficiency of m2PM imaging in freely moving animals. Firstly, perform virus injection and surgical procedures on the animals, such as cranial window implantation or GRIN Lens implantation, and then wait for 2-4 weeks to ensure full virus expression and surgical injury recovery. Taking a certain experiment as an example, we labeled GCaMP6s and mCherry in the primary motor cortex (M1) neurons, and then fixed the mice on a treadmill under the WF Nav device. Then locate the target area of interest using a wide field microscope and align it with the center of the field of view. Subsequently, switch to m2PM mode through the adapter and perform dual color two-photon imaging at wavelengths of 920nm and 1030nm. Finally, fine adjustments are made to the XY axis and focal plane to confirm the final target area within a few minutes. In most cases, the target focal plane is raised by about 25 µ m to compensate for the focal shift caused by cement shrinkage, and then the Baseplate is fixed on the animal's head piece. After about 10 minutes of curing, the mouse can perform free movement imaging.
Figure 6. Standardized free movement micro two-photon microscopy imaging process
The developers validated the system by imaging the calcium signals and structure of neurons in a mouse social experiment. The entire installation process took about 20 minutes, with a success rate of up to 100% (n=15). Even inexperienced researchers can easily master it, greatly improving experimental efficiency. In addition, multiple days of repeated imaging were performed on the neurons and astrocytes in the second layer of the M1 region, and the results showed stable field registration without reinstalling the Baseplate.
Figure 7. Multi day imaging with the same field of view
Thanks to standardized mechanical design, the m3PM imaging head can be conveniently installed on the bracket. By using a multifunctional adapter, seamless switching from wide field mode to three photon imaging mode can be achieved. Due to the limited imaging depth of wide field single photon microscopy, deep signals are often difficult to detect; Although superficial blood vessels can serve as a reference, preserving the virus injection area on the surface can provide more direct fluorescent labeling, which helps align deeper areas on the vertical axis. The developers successfully recorded neural activity in the postsubuculum area up to 1020 μ m deep from the cortical surface in mice with fixed head and GCaMP6f expression, and conducted open field behavior exploration experiments in mice, recording long-term calcium signal activity at a depth of 670 μ m.
Figure 8. Miniature Three Photon Fixed and Free Behavior Imaging
This work was completed by Professor Wu Runlong (corresponding author), Professor Wang Aimin, Professor Cheng Heping, and others from Beijing University of Information Science and Technology.
[Reference]
Wu R, Sun Y, Hao Z, et al. Wide-field fluorescence navigation system for efficient miniature multiphoton imaging in freely behaving animals. Neurophotonics. 2025; 12(2):025018. doi:10.1117/1.NPh.12.2.025018
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