BEIJING, Aug. 22 (Xinhua) — Chinese scientists have, for the first time in the world, achieved high-resolution deep-brain multicolor two-photon imaging in freely moving mice using a newly developed miniature two-photon microscope.
The study, which provides a new tool for decoding complex brain function mechanisms, was published online in the journal Nature Methods on Thursday.
The brain operates through the coordinated efforts of tens of billions of neurons and hundreds of trillions of synapses. Accurately capturing the dynamic changes in neuronal and synaptic activity has long been a major challenge in brain science research.
Two-photon microscopic imaging, a nonlinear optical imaging technique based on two-photon absorption and fluorescence excitation, offers high resolution and deep imaging capabilities.
In 2017, a research team led by Cheng Heping, director of the National Biomedical Imaging Center at Peking University (PKU), successfully developed China’s first-generation miniature two-photon microscope, achieving for the first time clear and stable functional imaging of synapses in freely moving mice.
One of the key components of the miniature two-photon microscope is a hollow-core fiber. Previously, hollow-core fiber could only transmit ultrafast laser pulses at a single wavelength, limiting its multicolor imaging capabilities.
A PKU research team led by Cheng and Wang Aimin, in collaboration with a research team led by Wu Runlong from Beijing Information Science and Technology University, developed a novel ultra-broadband hollow-core fiber.
This fiber, characterized by low loss and low dispersion, allows the transmission of femtosecond pulsed lasers at multiple wavelengths ranging from 700 to 1,060 nanometers. This innovation led to the creation of the multicolor miniature two-photon microscope weighing only 2.6 grams.
By placing this microscope on the heads of mice with Alzheimer’s disease, researchers simultaneously captured dynamic three-color images — red, green and blue — of neuronal calcium signals, mitochondrial calcium signals and plaque deposits. They observed abnormal cellular and mitochondrial activities near plaques even in the early stages of the disease.
“This is like a live color broadcast of the dynamic activities of neurons and organelles in the brain,” Wu said. In the past, due to the limitations of hollow-core fiber, microscopes could only observe single cell types. Now, with different cell types labeled with fluorescent markers of different colors, researchers can clearly observe complex interactions among multiple cell types and study how they coordinate and interact.
The researchers also obtained neuronal calcium signals and structural imaging in the mouse cerebral cortex at depths exceeding 820 micrometers, the deepest imaging currently known, achieved through a miniature two-photon microscope without damaging brain tissue.
Additionally, the microscope lens enables seamless switching between large-field observation and high-resolution fine imaging. With just a 30-second adjustment, the on-screen image can transition from a “micro close-up” to a “wide-angle panoramic view.”
The researchers overcame the challenge of multicolor excitation imaging in miniature two-photon microscopes, marking a breakthrough in the study of complex brain networks, Cheng said.
The new-generation microscope successfully achieves multicolor, deep-brain and cross-scale neural imaging in freely moving mice, with broad application prospects in understanding brain cognition principles, studying brain disease mechanisms, evaluating neuropharmaceuticals and developing brain-computer interfaces, he added. ■