 标题 1 True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. 亚细胞动态生理成像要求的是在细胞的母体组织里来研究它们，在那里会有各种各样的环境诱因，这些诱因会驱动基因表达，从而影响我们观察到的表现型。 A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. 想要全面进行理解还需要高时空分辨率的细胞和周围环境的容积成像图片，并且不会对细胞核周围环境带来不必要的压力。 We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. 我们采用了可调节的光学的晶格光片照明显微镜，来对大片多细胞容积进行观察，得到了亚细胞过程的无创无干扰图像，这些亚细胞过程包括内吞作用，有丝分裂期间的细胞器重构以及体内转移的癌细胞。 The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments. 这项技术揭示了不同组织的细胞内的表型多样性和发育过程，还可能为细胞是如何利用自身多样性来适应不同生理环境的提供线索。 标题 2 Introduction引言 Organisms live by means of the complex, dynamic, three-dimensional (3D) interplay between millions of components, from the molecular to the multicellular. 生物组织的存活依赖着上百万的各种组分之间的复杂，动态，三维的相互作用，既有分子水平的，也有多细胞的。 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. 想要看到这种复杂过程的原始形态要求在组织内部的各处进行高时空分辨率的成像，因为只有在这里才是那些所有调控细胞生理的环境因子存在的地方。 However, the optical heterogeneity of multicellular systems leads to aberrations that quickly compromise resolution, signal, and contrast with increasing imaging depth. 然而，多细胞系统光学差异性会导致偏差，会随着成像深度的增加而迅速降低分辨率，信号和对比度。 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. 而且，就算没有偏差，高分辨率和快速成像往往伴随着很强的照明，这回扰乱亚细胞的微妙过程，甚至带来永久性的光中毒效应。 标题 3 RATIONALE基本原理 We combined two imaging technologies to address these problems. 我们采用了两种成像技术来解决这个问题。 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. 首先，晶格光片显微镜（LLSM）迅速地重复扫过所观测容积内的超薄光片，同时获得一系列的图片，建立起高分辨率的内部动态3D影像。 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. 将照明限制在薄片内可以保证该范围外的其他区域不被曝光，而光片内荧光的实时收集会带来更低的，更少的扰动强度。 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. 第二种技术是可调节光学技术，通过测量样品对观察体积内创建的荧光“引导星”的扰动—这种扰动也会影响获得的光片成像—然后再通过改变镜子的形状来创造出一个等大反向的扰动，这样来进行补偿。 标题 4 RESULTS结果 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. 我们利用AO-LLSM技术在很宽的长度尺度范围内研究了不同的体内3D亚细胞过程，从纳米水平的网格蛋白小窝散射到200微米长度的发育中的斑马鱼的脊髓的轴向移动。 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. 细胞膜的清晰轮廓可以让我们从计算上分离并单独研究完整机体内复杂的多细胞环境里的任何想要研究的细胞。 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. 利用此法，我们可以对比不同细胞类型之间的特殊过程，比如肌肉纤维和脑细胞中的网格蛋白小窝内吞，大脑和眼部发育过程中的细胞分裂时的细胞器重构，以及免疫细胞和可转移乳腺癌细胞的移动机制。 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. 尽管大部分样品取自斑马鱼胚胎，我们还利用AO-LLSM研究了人类干细胞培养出的组织体，秀丽隐杆线虫和拟南芥叶片。 标题 5 CONCLUSION结论 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. AO-LLSM技术可以得到亚细胞过程高分辨率的活体细胞图像，从盖玻片的限制发展到了透明组织内更为接近生理的3D环境里。 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. 这就创建出了一种新的方法来研究细胞间动态，细胞外交流的表型多样性，以及不同细胞类型、生物体和发育阶段的集体细胞行为。 End Observing the cell in its native state: Imaging subcellular dynamics in multicellular organisms， Tsung-Li Liu1,*, Srigokul Upadhyayula1,2,3,4,*, Daniel E. Milkie1, Ved Singh1, Kai Wang1,†, Ian A. Swinburne5, Kishore R Science 20 Apr 2018: 感谢关注 跟amber一起看世界 主人太懒 不定期兼间歇性更新 那也不妨碍我们做个好朋友 |
|
|
