Miniature Multiphoton Microscopy Illuminates the Brain: Pioneering Deep-Tissue Imaging for Neuroscience

On February 23, 2023, the team of Cheng Heping and Wang Aimin from Peking University published an article online in Nature Methods titled “Miniature three-photon microscopy maximized for scattered fluorescence collection.”This paper reports a miniaturized three-photon microscope weighing only 2.17 grams, which achieves functional imaging of neurons across the entire cerebral cortex and hippocampus in freely behaving mice for the first time, thereby opening a new research paradigm for elucidating neural mechanisms within deep brain structures.

The hippocampus is located beneath the cortex and the corpus callosum, playing a crucial role in the consolidation of short-term memory into long-term memory, spatial memory, and emotional encoding. In rodent research models, the hippocampus lies at a depth greater than one millimeter from the brain surface. Due to the high light-scattering optical properties of brain tissue, particularly the corpus callosum, overcoming the imaging depth limit has long been a significant challenge for neuroscientists. Previous miniaturized single-photon and multi-photon microscopes have been unable to achieve non-invasive imaging of the hippocampal region by penetrating through the entire cortex.

Because brain tissue is a highly scattering medium, fluorescent signals originating from deep brain regions undergo random scattering before reaching the surface. Benchtop three-photon microscopes employ large-diameter objective lenses with a working distance greater than 2 mm and a numerical aperture of 1.0, thereby maintaining sufficient fluorescence signal collection efficiency while meeting the requirements for deep-tissue imaging. However, in miniature probes, using a high-numerical-aperture objective lens with a 2-mm working distance results in excessive probe weight and size, failing to meet the constraints on weight and volume required for freely behaving mice.

Therefore, the key to addressing this issue isMaintaining Sufficient Fluorescence Collection Efficiency While Reducing the Numerical Aperture of the Objective Lens。

The development of this miniaturized three-photon microscope introduces the classical Abbe condenser structure into the probe’s optical design. A miniature Abbe condenser is closely coupled with a simplified infinity-corrected objective to enhance the collection efficiency of scattered fluorescence. Additionally, a Lister objective is incorporated into the excitation path as a tube lens to compensate for aberrations in the simplified objective. Partial sharing of optical elements between the Abbe condenser and the Lister tube lens further reduces the probe’s volume. This new miniaturized microscope configuration achieves fluorescence collection efficiency comparable to that of benchtop three-photon microscopes, with a working distance of 1.75 mm, a numerical aperture of 0.65, and an objective diameter of only 3.4 mm.

Globally,This miniaturized three-photon microscope achieves, for the first time, a non-invasive deep-brain imaging solution for freely behaving animals. It can penetrate the entire cerebral cortex and corpus callosum of the mouse brain to enable direct observation and recording of the hippocampal CA1 subregion, thereby avoiding brain tissue damage associated with implanted GRIN lenses.The maximum imaging depth for neuronal calcium signals can reach 1.2 mm, while vascular imaging can achieve a depth of 1.4 mm. Meanwhile, the miniaturized three-photon microscope requires only a few milliwatts for whole-cortex calcium signal imaging and 20–50 mW for hippocampal calcium signal imaging, which is significantly below the safety threshold for tissue damage.

In the field of multiphoton imaging, this team from Peking University has been deeply engaged for many years.

2017,Led by Academician Cheng Heping, the Peking University interdisciplinary team successfully developed the first-generation 2.2-gram miniaturized two-photon microscope, FHIRM-TPM, achieving for the first time globally high-speed, high-resolution real-time imaging of neuronal functional activity at the level of individual dendritic spines in freely moving mice.This miniaturized two-photon microscope enables real-time recording of neuronal and dendritic spine activity in the brains of freely behaving animals, supports calcium imaging, and allows for long-term, repeated imaging within the same field of view.

Previously, when using benchtop two-photon microscopes to study brain activity in live mice, it was necessary to anesthetize the animals and immobilize their heads. Under these conditions, the mice could not move freely, making it impossible to study behaviors such as fighting, nursing, and social interaction, while research involving the tail suspension test and electric shock tests was limited. Furthermore, benchtop two-photon microscopes are inconvenient to move and occupy a large footprint.

With the advancement of technology, some researchers have introduced virtual reality (VR) techniques by placing a monitor in front of mice and positioning them on treadmills. During running, virtual scenarios are presented to observe brain activity in the mice. However, this approach remains highly controversial. First, neuronal responses in the mouse brain may not be entirely identical between virtual and real-world environments. Second, head-fixed animals experience emotional stress and may exhibit stress responses.

According to Wu Runlong, the miniaturized two-photon microscope developed by the Peking University team has achieved breakthroughs in several key areas, first,Flexible Transmission of Femtosecond LasersConventional optical fibers exhibit strong dispersion and nonlinear effects when transmitting femtosecond lasers, resulting in reduced two-photon excitation efficiency. The Peking University team has developed a novel hollow-core photonic crystal fiber capable of distortion-free transmission of 920 nm femtosecond laser pulses, thereby enabling efficient two-photon excitation of green fluorescent protein.

Second,Imaging Objective Lens, traditional objective lenses have a diameter of approximately 20 mm and weigh more than an average mouse; the miniature objective lens developed by the Peking University team has a diameter of 3.5 mm and a length of 10 mm, incorporates more than ten lens elements internally, and still achieves diffraction-limited resolution; third,High-Speed Imaging, to reduce motion artifacts, rapid imaging is required. The Peking University team achieved video-rate imaging speed in a miniaturized format using high-speed MEMS scanning galvanometers. Finally, the high-quality integration of various technologies resulted in a 2.2 g miniaturized two-photon probe with high spatiotemporal resolution.

Globally, this miniaturized two-photon microscope, designed to be worn while running, has achieved clear and stable imaging in freely behaving animals for the first time. It enables long-term observation of multiscale, multilevel dynamic changes in neural synapses, neurons, neural networks, and remotely connected brain regions under natural behavioral conditions—such as foraging, platform jumping, fighting, playing, and sleeping—or across pre-learning, during-learning, and post-learning phases, thereby capturing dynamic images of neuronal and synaptic activity in the brains of mice during free behavior.

Dr. Edvard I. Moser, a Nobel laureate in Physiology or Medicine, has described this microscope as a “revolutionary” new tool in the field of neuroscience research.

In 2021, this Peking University team launched the second-generation miniaturized two-photon microscope, FHIRM-TPM 2.0, which expanded the field of view by 7.8-fold and improved lateral and axial resolutions by approximately 1.5 times compared to the first-generation model, while also enabling three-dimensional imaging capable of capturing functional signals from thousands of neurons in the cerebral cortex.

In addition to upgrading core performance,The new generation of miniaturized two-photon microscopes adopts an integrated all-in-one design, meeting the application needs of laboratories with limited space. It also features strong compatibility, with a built-in laser adaptation module that can match femtosecond lasers from all brands on the market. With more powerful imaging performance, broader adaptability, and easier operation, it brings new vitality to the development of imaging technology.

Based on miniaturized two-photon microscopes, the Peking University team has collaborated with Hu Hailan’s team at Zhejiang University, Sun Yangang’s team at the Chinese Academy of Sciences, and others. For instance, Sun Yangang’s team is investigating the neural mechanisms underlying itch perception. Since the onset of itch occurs rapidly and neuronal firing rates are high, with different subsets of neurons activated at distinct stages of itch perception, the imaging equipment must not only capture individual neurons at high speed but also possess high spatial resolution to distinguish between these two types of neurons.

Previously, studies on the coding mechanisms of itch perception in the cerebral cortex were mostly conducted in anesthetized animals, which precluded the reporting of itch perception through scratching behavior. By using miniaturized two-photon microscopes, Sun Yangang’s team achieved calcium imaging at single-cell resolution in freely moving mice, providing a new approach for studying the mechanisms of itch.

In terms of application scenarios, the Peking University team has also been pushing the boundaries. In 2022, the team overcame multiple technical challenges, including organismal stress responses and protection in the extreme environments of spaceflight, and developed a two-photon microscope for the space station. As previously introduced by Academician Cheng Heping, “On-orbit experimental instruments and equipment face more stringent requirements for reliability, size, weight, and resistance to shock and vibration. Developing a two-photon microscope capable of operating in space is no easy feat.”

Recently, the Shenzhou-15 astronauts successfully acquired three-dimensional images of the epidermis and superficial dermis using the two-photon microscope aboard the space station, which can be utilized for in-orbit health monitoring of the crew.

Wang Aimin pointed out that, due to comprehensive technical challenges and other factors, only a few research groups worldwide are currently studying miniaturized two-photon microscopes. It is only by attracting more participants that the technology can be promoted more rapidly and the industry expanded.

The large-scale promotion of innovative technologies is inseparable from industrialization. Beijing Chaoweijing Biological Technology Co., Ltd. (hereinafter referred to as “TRANSCEND VIVOSCOPE”), established with the backing of academicians of the Chinese Academy of Sciences and multidisciplinary experts from Peking University in biology, medicine, physics, and physiology, has successfully achieved the industrial translation of scientific research outcomes. Currently, TRANSCEND VIVOSCOPE’s teams in Beijing and Nanjing have grown to over 100 members, attracting industry professionals with extensive experience in production, regulatory registration, and market promotion.

From research projects to industrialization, in addition to a company’s own technological accumulation, differentiated product innovation, and process control, a controllable supply chain is one of the key factors.

According to Wang Aimin, the first project we undertook as a “commercial pilot” was a two-photon light-sheet microscope. One of its core components, the zoom lens, was supplied by only one U.S. company. While we were able to purchase it without issue during the research and small-scale application phases, the supplier ceased providing it once we moved toward industrialization, directly leading to the project’s premature termination.

In the subsequent industrialization process, TRANSCEND VIVOSCOPE adopted a model combining “self-reliance” with “internal and external synergy.” First, it independently developed technologies to gradually localize the core components of its two-photon microscope products. Second, it collaborated with domestic industry stakeholders, who developed new technologies based on specific demands.

Currently,TRANSCEND VIVOSCOPE has established a controllable industrial chain, with key components such as femtosecond lasers, hollow-core fibers, and miniaturized objectives now domestically produced in China. This lays a solid foundation for the future development of the company’s medical product lines, enabling autonomy and control in R&D and manufacturing while significantly reducing production costs.。

Wang Aimin stated, “China’s brain science research started relatively late, with a market size less than one-tenth that of the United States. However, the China Brain Project was officially launched in 2021, with an estimated investment of RMB 50 billion over five years.” Meanwhile, two-photon microscopy has been applied to in vivo pathological research and applications globally for nearly two decades. As a powerful tool for live imaging, two-photon microscopy has also demonstrated significant potential in clinical diagnostic research. TRANSCEND VIVOSCOPE adheres to a dual-track strategy, developing both scientific research equipment and medical devices, striving to translate findings from animals to humans and thereby contribute more substantially to human health.

The handheld skin biological cell detector is the company’s first “foray.”This product is based on miniaturized two-photon microscopy technology, enabling in vivo, in situ, non-invasive, and label-free micro- and nano-scale microscopic imaging., cells, elastic fibers, collagen fibers, metabolic information, and other features are clearly visible.

Handheld skin biological cell detectors mainly have two major application markets. One is clinical application, conducted within hospitalsReal-time in vivo detectionCurrently, the gold standard for in-hospital disease diagnosis is pathological examination, which requires excising a small tissue sample from the human body for analysis. This procedure is invasive and typically entails a waiting period of 3–5 days to obtain results. However, many clinical scenarios demand rapid decision-making and regular monitoring, such as routine follow-ups for melanoma and skin cancer. With handheld skin cellular bioscopes, physicians can monitor changes in cell morphology in real time, assess disease progression, and adjust treatment plans accordingly.

Second,Skin Efficacy TestingTRANSCEND VIVOSCOPE is conducting research on skin atlases, which can be used to evaluate cosmetics and medical aesthetic technologies. According to Wang Aimin, while cosmetics claim to help improve skin condition, there are no objective standards in place. By directly observing skin cells and detecting elastic fibers and collagen fibers, we can establish an objective standard for skin age to assess the efficacy of cosmetic products.

In the medical field, TRANSCEND VIVOSCOPE is developing 3D 4K fluorescence endoscopes that incorporate cellular imaging capabilities into traditional endoscopy, enabling physicians to visualize more information and further advancing precise diagnosis.

Wang Aimin stated, “Market education is crucial for the adoption of new technologies, particularly in the conservative and cautious healthcare market. We must first thoroughly understand physicians’ usage habits before designing products, ensuring that innovative technologies are transformed into tools that are truly user-friendly for clinicians.” Currently, TRANSCEND VIVOSCOPE is collaborating with the Chinese PLA General Hospital (301 Hospital) to develop single-use endoscopes for the screening and diagnosis of early-stage gastrointestinal cancers. The company is also conducting joint research with the Department of Obstetrics and Gynecology at Peking Union Medical College Hospital on early screening for cervical cancer.

Brain science is undoubtedly one of the hottest topics in the medical field in recent years.

The human brain features an intricate structure comprising tens of billions of neurons, and scientists are still exploring the mechanisms underlying emotions and feelings. Imaging technology is pivotal to the advancement of brain science. From CT and MRI to PET, these imaging modalities all aim to achieve “seeing is believing.” Researchers continually strive to perform high-resolution imaging directly on fresh samples and even in living organisms. Multiphoton microscopy offers significant advantages in terms of resolution, speed, and imaging depth, thereby empowering brain science research.

Furthermore, it will further elucidate the pathogenesis of brain diseases and guide the development of diagnostic and therapeutic methods. Neurological disorders such as epilepsy, Parkinson’s disease, and Alzheimer’s disease impose substantial social and economic burdens that persist over long periods, while effective treatment options remain limited. New insights into disease mechanisms could enable early prevention and intervention, thereby significantly alleviating the societal burden.

Furthermore, imaging technology will inevitably be closely integrated with digital technologies, particularly artificial intelligence (AI), and “AI+” imaging systems will further enhance clinicians’ diagnostic and therapeutic capabilities.

In summary, multiphoton microscopy boasts broad application potential in both research and clinical settings, serving as a lighthouse in the vast expanse of brain science to illuminate many hidden corners.

Reference Article:

"Why Do You Feel Itchy? CAS Research Reveals Neural Mechanisms Underlying the Representation and Perception of Itch" — The Paper

"[Frontiers in Science and Technology] Peking University Team Led by Cheng Heping and Wang Aimin Successfully Develops Miniaturized Three-Photon Microscope" — Chinese Society for Biophysics

“Application of Multiphoton Microscopy Imaging Technology in the Study of Brain Diseases” — Huang Yanxia, Zhou Feifan, Zhou Ting, Xu Hao, Lin Danying, Qu Junle

《Miniature three-photon microscopy maximized for scattered fluorescence collection 》——《Nature Methods》

“A World First! Peking University Develops Space Station Two-Photon Microscope to Capture 3D Images of Astronauts’ Skin” — Peking University