The unremitting pursuit of high-speed, high-resolution long-term observation ability of living organisms has run through the development history of microscopes for centuries. It is an icebreaker in life science and medical research, and a pioneer in discovering new phenomena and revealing new mechanisms. However, due to the limitation of three-dimensional tissue distribution, optical aberration, phototoxicity and many other adhesion problems, the long-term observation of high-speed subcellular resolution in mammalian living environment has not been solved, which greatly restricts the in-depth study of brain science, oncology and immunology.
On may 25th, 20021,the research group of Dai Qionghai, academician of Li Yu's Department of Automation, and the research group of the School of Life Sciences, Tsinghua University Institute of Brain and Cognitive Sciences, published a research paper entitled "Digital adaptive optical iterative photography allows the in vivo observation of three-dimensional cellular dynamics in milli-inch scale on cells for one hour". The team creatively proposed the digital adaptive optical framework and invented the scanning light field imaging technology. After three years of research, they developed a scanning light field microscope, collectively known as (DAOS limit). In the imaging field of view of 225 22516 μ m3, with the optical diffraction limit resolution of 220nm in the transverse direction and 400nm in the axial direction, DAOSLIMIT will increase the three-dimensional continuous observation time of living body from several minutes to several hours, improve the spatio-temporal resolution of living body imaging by two orders of magnitude, and reduce the phototoxicity by three orders of magnitude, which provides a brand-new path for revealing the interaction between multi-cells and multi-organelles in mammals.
Although the traditional light field microscope can record the multi-angle information of the sample at a time and realize fast three-dimensional imaging, the spatial resolution and angular resolution cannot be taken into account. Through the introduction of small-scale vibration of high-speed galvanometer and spatial superposition constraint, DAOSLIMIT can obtain high-resolution spatial and angular four-dimensional all-optical field information with full photon efficiency, thus realizing incoherent aperture synthesis, and then avoiding the loss of time resolution caused by scanning by using the spatial-temporal continuity prior of the sample.
A bottleneck restricting the observation of living tissue is the serious optical aberration caused by the different three-dimensional spatial distribution of sample refractive index, which significantly reduces the spatial resolution of microscopic imaging. The traditional adaptive optical microscope, which originated from astronomical observation, can only realize aberration correction imaging in a small field of view even through complex software and hardware. DAOSLIMIT establishes a new framework for digital adaptive optics (DAO) imaging, which does not need additional wavefront sensor or spatial light modulator. By obtaining four-dimensional all-optical information, signal acquisition and adaptive wavefront correction are decoupled, and adaptive optical correction in a large range of spatial blocks is realized in the post-processing process, which improves the spatial resolution to the optical diffraction limit.
Phototoxicity caused by light is another long-term pain point of in vivo fluorescence imaging. Due to the non-imaging laser irradiation area in the rotating disc focusing microscope (SDCM) and two-photon microscope (two-photon microscope), high-speed shooting for a few minutes will bring serious photobleaching/photodamage effect to the samples. Optical microscope (LSM) can alleviate this problem by exciting only the focal region, but it cannot maintain subcellular resolution in opaque tissues. DAOSLIMIT adopts the mode of simultaneous excitation and acquisition in the same three-dimensional area, and makes full use of each photon to image by expanding the axial depth of field. Compared with the conventional microscope, only μW excitation light intensity is needed to obtain enough signal-to-noise ratio, and the phototoxicity is reduced by three orders of magnitude. For the first time in the world, millisecond continuous high-speed observation of mammals for several hours has been realized.
Migrasome is a new organelle recently discovered and named by Li Yu's laboratory. It is now known that migra some plays an important role in embryonic development and the maintenance of immune system. With the help of DAOSLIMIT, a new field of research on the function of migratory bodies in the living environment of mammals can be created. The researchers stained neutrophils and blood vessels respectively, and performed multicolor imaging in the liver of living mice. For the first time, the formation and changes of mammalian migratory bodies and filamentous pseudopods were clearly observed. "through DAOSLIMIT, we observed that immune cells leave many migrants when they move in the blood vessels of the living mouse liver. It is possible for immune cells to realize large-scale information exchange and long-distance interaction between cells by producing migratory bodies, which may be a new mechanism. These immigrants, like the beacon tower of the Great Wall, may play a key role in transmitting signals in a series of complex reactions such as immune monitoring. " Professor Li Yu said.
The researchers further injected human tumor cells into the living zebrafish larvae. With the extremely weak phototoxicity of DAOSLIMIT, a new phenomenon that tumor cells actively adapt to the environment through vesicles and filamentous structures was discovered in continuous high-speed and long-term observation. Professor Dai Qionghai pointed out, "The new instrument technology provides a new path for life science and medical research, and these interesting new phenomena are just the tip of the iceberg. With the advancement of technology, the discovery of more new phenomena and the revelation of new mechanisms in the future are expected to help new breakthroughs in major basic research related to people's lives and health, such as brain science, cancer and immunity. "
Wu Jiamin, a postdoctoral fellow in the Department of Automation of Tsinghua University, Lu Zhi, a doctoral student, and Jiang Dong, a postdoctoral fellow in the School of Life Sciences, are the first authors of the thesis, and Dai Qionghai, a professor in the Department of Automation of Tsinghua University, Beijing National Information Science and Technology Research Center, Institute of Brain and Cognitive Science, and Fan Jingtao, an associate researcher. Professor Li Yu from the School of Life Sciences is the correspondent of this article. It is reported that related technologies have been transformed.
Original link:
https://doi . org/ 10. 10 16/j . cell . 202 1.04 . 029
Producer: Eleven
refer to
[1] max C. "adaptive optics and its history" the197th meeting of the American astronomical society. NSF Adaptive Optics Center at the University of California, Santa Cruz and DOE Lawrence Livermore National Laboratory. (200 1).
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