诺奖得主Science发表突破成果:癌细胞移动、神经发育,新技术都能“看得到”
2018/04/22
因“开发出超分辨率荧光显微镜”获得2014年诺贝尔化学奖的Eric Betzig博士又取得了一项突破成果。由他带领的团队最新开发出了一款结合2种成像技术的显微镜,可使研究人员观察活细胞前所未有的3D细节,包括癌细胞移动、脊髓神经回路连接以及免疫细胞在斑马鱼内耳中游走等。


在斑马鱼胚胎的脊髓中,新的神经元以不同的颜色发光,让科学家能够追踪神经回路的发育Credit: T. Liu et al./Science 2018

4月20日,这项成果以 “Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms”为题发表在Science杂志上。作者们认为,新技术解决了在活体组织中进行细胞成像这一长期存在的问题,为生物学研究提供了令人振奋的新视角。


图片来源:Science

克服传统显微镜的障碍

为了获得清晰的图像,传统的显微镜通常会将他们的实验对象隔离在一个载玻片上,或者用潜在的有害数量的光(harmful amounts of light)来照射它们。但Betzig博士认为,观察载玻片上被隔离的细胞就好像到动物园去研究狮子的行为一样(并不能看到细胞在原始环境中的真实行为)。

An immune cell migrates through a zebrafish's inner ear while scooping up particles of sugar (blue) along the way. Credit: T. Liu et al./Science 2018(视频地址:https://v.qq.com/x/page/a0633e8uelf.html)

为了克服这些障碍,Betzig博士和他的团队结合了两个他们于2014年首次报道的显微镜技术。现在,利用这一新装置,研究人员能够在细胞所处的自然环境下观察它们(而不是将它们分离、独立出来再观察它们)。上面的视频展示了在斑马鱼胚胎内耳中移动的一个免疫细胞。


Imaging cellular diversity in a developing zebrafish(图片来源:Science)

新技术的两大改进

第一步:让细胞“活着”

为了制作这个免疫细胞视频,Betzig博士及其同事避开了传统显微镜使用的强烈光线,因为这种光线会破坏或杀死活细胞。相反,研究小组使用了一种被称为“lattice light-sheet microscopy”的技术,该技术能够使一层薄光(a thin sheet of light)以非常高的速度不断地穿过活体组织,从而将细胞损伤降至最低水平,同时获得一系列2D图像,构建亚细胞动力学的高分辨3D电影(building a high-resolution 3-D movie of subcellular dynamics)。

第二步:使细胞周围环境不被“扭曲”

同时,为了使细胞的周围环境不被“扭曲”,研究人员使用了自适应光学adaptive optics,天文学家使用的一种成像技术)。该技术能够帮助解决“扭曲”问题,并校正图像。


图片来源: Howard Hughes Medical Institute

Betzig博士说:“如果没有自适应光学,所有这些细节都很难看到。”在他看来,自适应光学是当今显微镜研究中最重要的领域之一,而擅长3D活体成像的“lattice light sheet microscope”则是展现其力量的完美平台。同时,他还指出,目前自适应光学还没有真正“起飞”,因为这一技术复杂且昂贵,但是未来10年内,世界各地的生物学家都将参与其中。

前所未有的3D分辨率

借助结合了“lattice light-sheet microscopy”和“自适应光学”的这一新显微镜技术,研究人员现在能够窥视生物体的内部,以前所未有的3D分辨率观察细胞间的相互作用。以下是9张酷炫的动图:


人类干细胞衍生类器官中的内吞作用


早期斑马鱼大脑中的细胞器动力学


斑马鱼眼睛中的细胞器动力学


网格蛋白介导的体内内吞作用


斑马鱼眼睛中膜动力学


脊髓神经回路发育的体内成像


网格蛋白在肌肉纤维中的定位


对大体积成像进行“Tiled Adaptive Optics”校正


斑马鱼异种移植模型中癌细胞的迁移

下一步计划

最后,值得一提的是,这一装置目前需要一个3米长的桌子,Betzig博士等正致力于让其更小巧、更人性化。“评判一个显微镜价值的唯一标准是,有多少人能使用它,以及人们用它所发现的东西的重要性。”他说。最终,Betzig博士希望这一新技术能够被商业化,让自适应光学成为主流。

责编:风铃

参考资料:

Cutting-edge microscope spies on living cells inside the body

New Microscope Captures Detailed 3-D Movies of Cells Deep Within Living Systems

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  • Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms

    INTRODUCTION Organisms live by means of the complex, dynamic, three-dimensional (3D) interplay between millions of components, from the molecular to the multicellular. Visualizing this complexity in its native form requires imaging at high resolution in space and time anywhere within the organism itself, because only there are all the environmental factors that regulate its physiology present. However, the optical heterogeneity of multicellular systems leads to aberrations that quickly compromise resolution, signal, and contrast with increasing imaging depth. Furthermore, even in the absence of aberrations, high resolution and fast imaging are usually accompanied by intense illumination, which can perturb delicate subcellular processes or even introduce permanent phototoxic effects. RATIONALE We combined two imaging technologies to address these problems. The first, lattice light-sheet microscopy (LLSM), rapidly and repeatedly sweeps an ultrathin sheet of light through a volume of interest while acquiring a series of images, building a high-resolution 3D movie of the dynamics within. The confinement of the illumination to a thin plane insures that regions outside the volume remain unexposed, while the parallel collection of fluorescence from across the plane permits low, less perturbative intensities to be used. The second technology, adaptive optics (AO), measures sample-induced distortions to the image of a fluorescent “guide star” created within the volume—distortions that also affect the acquired light-sheet images—and compensates for these by changing the shape of a mirror to create an equal but opposite distortion. RESULTS We applied AO-LLSM to study a variety of 3D subcellular processes in vivo over a broad range of length scales, from the nanoscale diffusion of clathrin-coated pits (CCPs) to axon-guided motility across 200 μm of the developing zebrafish spinal cord. Clear delineation of cell membranes allowed us to computationally isolate and individually study any desired cell within the crowded multicellular environment of the intact organism. By doing so, we could compare specific processes across different cell types, such as rates of CCP internalization in muscle fibers and brain cells, organelle remodeling during cell division in the developing brain and eye, and motility mechanisms used by immune cells and metastatic breast cancer cells. Although most examples were taken from zebrafish embryos, we also demonstrated AO-LLSM in a human stem cell–derived organoid, a Caenorhabditis elegans nematode, and Arabidopsis thaliana leaves. CONCLUSION AO-LLSM takes high-resolution live-cell imaging of subcellular processes from the confines of the coverslip to the more physiologically relevant 3D environment within whole transparent organisms. This creates new opportunities to study the phenotypic diversity of intracellular dynamics, extracellular communication, and collective cell behavior across different cell types, organisms, and developmental stages.

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