Nat Biotechnol: A new light field microscope records rapid dynamic changes in brain neuron activity and blood flow at high speed.

On August 10, 2020, nature Biotechnology published a research paper online entitled "Rapid body imaging of the brains of mice and zebrafish by a confocus light field microscope", which was completed by the Research Group of Wang Kai, Center for Brain Science and Intelligent Technology Excellence of the Chinese Academy of Sciences (Neuroscience Research Institute), Shanghai Brain Science and Brain Research Center, and National Key Laboratory of Neuroscience.
the study developed a new type of body imaging technology: confocal light field microscopy, which allows rapid, large-scale body imaging of nerve and vascular networks in deep brain tissue in living animals.
DOI:doi.org/10.1038/s41587-020-0628-7 How large-scale neurons across the brain region integrate information and influence behavior is a central question in neuroscience, and answering this question requires tools to capture dynamic changes in the activity of a large number of neurons at higher space-time resolutions.
traditional tools for living brain imaging, such as convesonal microscopes and dual photon microscopes, are based on point scanning, and the time resolution is low, which is difficult to study the rapid changes of neurons in a wide range of brain regions.
, people have been working in recent years to develop faster imaging methods.
in a variety of new technologies, light field microscope has particular potential, has been widely concerned.
Its characteristic is that it can record signals from different depths of the object at a single exposure moment of the camera, reconstruct the entire three-dimensional body through anti-converge algorithm, achieve rapid body imaging, and has been applied to small pattern animals such as in-line worms and zebrafish.
two difficult problems in the traditional light field microscope, which limits its wide application in bioimaging.
first, the results of the refactoring are distorted.
The new augmented field-of-view light field microscope (eXtendedfield-of-view Light Field Microscopy, XLFM), developed by Wang Kai's research team in 2017, effectively solved this problem and was successfully applied to the full-brain neuron functional imaging of free-acting zebrafish larvae, recording for the first time three-dimensional changes in the activity of zebrafish larvae in total brain neuron activity during complete predatory behavior.
, existing light field microimaging techniques lack the optical secum to image thicker tissues, such as the brains of mice.
makes the optical field microscope have the same optical scugging capability as a confocus microscope, filtering out background signals other than the focal layer in large samples to improve the signal-to-noise ratio, is the key to improve the image quality, can be widely used.
, however, traditional convective microscopes use laser point-by-point scanning and conjugate point pinhole detection to reduce out-of-focus noise, which is not suitable for 3D optical field microscopes.
Faced with this challenge, the research team innovated the concept of general convection detection, which can be combined with the three-dimensional imaging strategy of the photostation microscope to effectively filter out background noise without sacrificing body imaging speed, and greatly improve sensitivity and resolution.
this new light field microimaging technology is called a confocused light field microscope.
1 (top) cofocus on the principle schematic of the light field microscope.
(lower) is different from traditional light field microscopes, confocused light field microscopes use flaky lighting, selective excitation of a portion of the sample, scanning in the direction of vertical illumination, the collected signal is filtered out of the focal layer by the shield.
reconstructs the captured image to obtain three-dimensional information within the focal layer.
team tested the imaging capabilities of confocused light field microscopes on different animal samples.
First, team members performed full-brain calcium imaging on buried live zebrafish larvae, comparing the results of confocus and traditional light field microscopes, and found that with the addition of optical sequestration capabilities, the image resolution and signal noise ratio increased significantly, allowing for more weak calcium activity.
further, a confocused light field microscope and a high-speed 3D tracking system are combined to perform full brain calcium imaging of free-acting zebrafish larvae, achieving a spatial resolution of 2 x 2 x 2.5 μm3 and a time resolution of 6Hz in a volume of 800 m x 200 m.
benefit from higher resolution and sensitivity to identify changes in the activity of calcium ions in individual neurons during herbivores.
example of the predatory behavior of zebrafish in Figure 2 (left).
0s is the moment a zebrafish devours grassworms.
zebrafish predaging behavior (right), a confocused light field microscope recorded neuron activity in two different brain regions.
arrow refers to a single neuron activated during the process.
, the team verified the imaging effect of a confocused light field microscope on the brains of mice, calcium imaging of the visual cortical layer of sober mice, and recorded the activity of nearly a thousand neurons in the volume of 800 m x 150 m at the same time, up to a depth of about 400 m, and steadily recorded more than 100,000 frames for more than 5 hours without significant light bleaching.
team members further attempted to image blood cells in the mouse brain using a confocused photostronic microscope at depths of up to 600 m and a shooting speed of 70 Hz, while recording the flow of group blood cells in thousands of blood vessel branches and calculating the speed of blood cells, which is more than a hundred times higher than the previous traditional imaging method.
network of blood vessels in the visual cortical layer of mice captured by a confocused light field microscope in Figure 3 (left).
six volumes shot at different depths are connected to a three-dimensional structure up to a depth of 600 m.
projection of a deep vascular network of 100 m to 250 m, the color represents the average flow rate of blood cells in different blood vessel branches.
blood vessel branches in the area indicated by the arrows in the figure (right) are counted for the number of blood cells that flow over a period of time.
team demonstrated higher resolution and sensitivity in the brains of free-acting zebrafish larvae and mice, providing new tools for studying the function of a wide range of neural and vascular networks.
addition, the technology is suitable not only for imaging brain tissue, but also for flexible resolution, imaging range and speed according to the type of sample required for imaging, and is used in fast dynamic imaging of other thick tissues.
The study was conducted under the guidance of Wang Kai researchers, mainly by doctoral students Zhang Qikun, Bai Wei and assistant researcher Jungle, Wang Kai Research Group Yu Peng, Zhang Tianlei, Chinese University of Science and Technology undergraduate Shi Wanzhu, DuJuLin Research Group Li Faning also made important contributions, DuJuLin researchers involved in cooperation and give guidance.
research has been strongly supported by the experimental animal platform of the Center of Brain And Intelligence Excellence of the Chinese Academy of Sciences.
research received project funding from the Ministry of Science and Technology of China, the Chinese Academy of Sciences, the National Natural Science Foundation of China and Shanghai.
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