Recently, Professor Wu Runlong's team from Beijing University of Information Science and Technology, in collaboration with Professor Xiao Shumin's team from Harbin Institute of Technology, Associate Professor Wang Aimin's team from Peking University, and Professor Feng Lishuang's team from Beihang University, successfully developed the second-generation composite super lens miniaturized two-photon microscope (Meta-m2PM 2.0), achieving high signal-to-noise ratio imaging of freely acting mouse neurons for the first time. The relevant results indicate that superlens are no longer just a laboratory concept, but can support high-quality brain functional imaging under natural animal behavior conditions, and contribute to the development of biomedical imaging. The research paper "Compound metal based miniature two-photon microscope for large FOV imaging in freely owned animals" was published online in PhotoniX (5-year average impact factor 19.3).

Screenshot of the online publication of the paper

Miniature two-photon microscopy has become an important tool in neuroscience research to analyze the activity of animal brain neurons in a state of free behavior. Currently, high-performance miniaturized two-photon microscopes have achieved breakthroughs such as high spatiotemporal resolution, large field of view, and multi-color imaging^[1-3]However, existing high-performance miniaturized two-photon microscopes still face the challenge of "fish and bear's paw cannot coexist" - it is difficult to balance high imaging performance with further lightweighting of the probe: their weight is generally greater than 2g (such as FHIRM-TPM weighing 2.15g)^[1]、FHIRM-TPM 2.0 重2.45g^[2]、FHIRM-TPM 3.0 重2.6g^[3]、 UCLA 2P Miniscope weighs approximately 4g^[4]）Therefore, it is difficult to apply to weight sensitive small animals such as songbirds, bats, young mice, and multi brain area observations of small animals.

Super lens technology provides the possibility to break through this bottleneck, and with its advantages of ultra-thin, high numerical aperture, and flexible design of aberration correction, it provides new ideas for miniaturized microscope optical systems. However, previous miniaturized two-photon microscopes based on a single hyper lens design were unable to solve the problems of limited field of view and large off-axis aberrations, greatly limiting their practical applications.

The project team designed a composite hyper lens architecture for "secondary imaging" (Figure 1a, b), decoupling scanning and focusing functions, breaking the balance between weight, field of view, and resolution in traditional miniaturized imaging architectures, and providing a new method for multi brain imaging of freely moving animals.

Figure 1 Structure and design of Meta-m2PM 2.0: (a) Schematic diagram of the composite metamaterial lens architecture for implementing a two-stage imaging system. D is the diameter of the incident laser beam; MEMS stands for Micro Electro Mechanical Systems; FOVimage is the field of view at the focal plane; Fscan is the focal length of the scanning lens; α is the MEMS tilt angle; θ is the MEMS biaxial scanning angle. (b) Outline and optical path diagram of Meta-m2PM 2.0 probe component. HC-920 is an air core fiber used for 920 nm transmission; SFB is a flexible fiber optic bundle

The joint team further designed polarization insensitive high aspect ratio Si for two-photon applications₃N₄The cellular library achieved dual band optimization of 920nm excitation light and 500-560nm fluorescence collection through the collaborative design of a double-layer hyper lens objective and a large angle hyper lens scanning mirror (Figure 2a, b), reducing the weight of the objective lens to one tenth of traditional miniaturized objectives and the probe weight to only 1.06g (Figure 2c, d). The new generation of composite super lens miniaturized two-photon microscope has achieved multiple breakthroughs: the field of view has been expanded to 350 × 330 µ m²More than 50 times higher than the first generation; The horizontal resolution at the center reaches 1.17 µ m, maintaining over 80% resolution even at the edge of the field of view; Moreover, the research team clearly captured the fine axonal structure of living mice and long-term activity imaging of neurons at a depth of 210 µ m (Figure 2e).

Figure 2: Preparation of Superlenses and Performance Testing of Probes: (a) Schematic Diagram of a Single Cell. H is the height (1500nm); L is the length (90-350nm); P is the period (400nm). (b) Scanning electron microscopy image of the prepared metamaterial lens: (i) front view; (ii) Central area; (iii) Edge region. (c) Photos taken with a meta lens objective (MO, weighing 0.05 g) and a FHIRM-TPM 3.0-U objective (3.0-U, weighing 0.5 g). (d) Photo of Meta-m2PM 2.0 probe component. (e) Left image: Structural imaging of M1 cortex in Thy1 YFPH transgenic mice with fixed head at a depth of 154 μ m. Right image: Calcium imaging of M1 cortical neurons expressing GCaMP6f in mice at a depth of 210 μ m

In the free behavior mouse brain imaging experiment, the research team stably tracked the calcium activity of over a hundred neurons, achieving a signal-to-noise ratio of 7.6 and being able to track the same batch of neurons for a long time (Figure 3-a-c). Thanks to the ultra lightweight probe design of 1.06g, the inertia effect of Meta-m2PM 2.0 is significantly reduced and stability is improved compared to previous miniaturized two-photon microscopes. In short-term stability tests, 90% of the field of view displacement is less than 5 µ m, and in long-term observations across the sky, the displacement does not exceed 10 µ m (Figure 3d, e).

Figure 3 Calcium imaging of free moving mice using Meta-m2PM 2.0: (a) Left: photo of mice wearing Meta-m2PM 2.0; Right: 12 minute mouse movement trajectory. (b) Left: Image at a depth of 190 μ m in the M1 cortex of freely moving mice; Right: Segmentation map of 122 neurons. (c) The signal-to-noise ratio (SNR) of neuronal calcium transients during a 12 minute recording period. (d) Imaging of the same field of view in the M1 cortex was performed on different days, with circles indicating the parts of the same neurons that were repeatedly identified in different experiments. (e) Short term and long-term stability of imaging. Left: Short term stability analysis of field of view displacement in the x and y directions in 8 experiments (>600s each) conducted by three mice under open field free exploration conditions; Right: Long term field of view displacement corresponding to the experiment in (d)

This research achievement has successfully achieved breakthroughs in ultra lightweight, large field of view, deep cortex, and high resolution by deeply integrating composite hyper lens technology with miniaturized two-photon microscopy imaging technology. Further practical application of hyper lens technology, achieving for the first time the imaging of free acting animal brain function using a hyper lens miniaturized two-photon microscope.

In the future, research on hyper lens technology will be further expanded to include multi-color imaging of free behavior, synchronous observation of multiple brain regions, and cross species (bats, birds, and juvenile rodents) studies in neural network analysis. It is expected to play an important role in the analysis of brain cognitive principles, early diagnosis of brain diseases, and evaluation of neural drugs.

The first authors of this paper are Hao Zeyu and Zhang Yao from Harbin Institute of Technology (Shenzhen), who were jointly trained by Beihang University and Beijing University of Information Science and Technology; The co corresponding authors are Professor Wu Runlong from Beijing University of Information Science and Technology, Professor Xiao Shumin from Harbin Institute of Technology, and Associate Professor Wang Aimin from Peking University. Professor Wu Runlong is responsible for the overall concept of the research project, leading the design and construction of the core optical system; Professor Xiao Shumin guides the design and optimization of superlenses; Professor Wang Aimin provided key technical support for the biological imaging experiment. This project has received funding support from the 2030 "Brain Science and Brain like Research" major project, the Beijing Laboratory of Biomedical Imaging, the National Natural Science Foundation of China, the Beijing Natural Science Foundation, and the Shenzhen Basic Research Project.

Hao, Z., Zhang, Y., Zhu, Y. et al. Compound metalens-based miniature two-photon microscope for large-FOV imaging in freely behaving animals. PhotoniX 6, 57 (2025).https://doi.org/10.1186/s43074-025-00218-y

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