Continuing the resolution revolution The living cell contains dynamic, spatially complex subassemblies that are sensitive to external perturbations. To minimize such perturbations, cells should be imaged in their native multicellular environments, under as gentle illumination as possible. However, achieving the spatiotemporal resolution needed to follow three-dimensional subcellular processes in detail under these conditions is challenging: Sample-induced aberrations degrade resolution and sensitivity, and high resolution usually requires intense excitation. Liu et al. combined noninvasive lattice light-sheet microscopy with aberration-correcting adaptive optics to study a variety of delicate subcellular events in vivo, including organelle remodeling during mitosis and growth cone dynamics during spinal cord development. Science, this issue p. eaaq1392 Adaptive optical lattice light-sheet microscopy permits delicate 3D subcellular processes to be viewed natively in vivo. 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. High-resolution in vivo cell biology. AO-LLSM permits the study of 3D subcellular processes in their native multicellular environments at high spatiotemporal resolution, including (clockwise from upper left) growth of spinal cord axons; cancer cell metastasis; collective cellular motion; endocytosis; microtubule displacements; immune cell migration; and (center) organelle dynamics. 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. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. 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. 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.

中文翻译：

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观察细胞的天然状态：对多细胞生物中的亚细胞动力学进行成像


继续分辨率革命活细胞包含动态的、空间复杂的子组件，对外部扰动敏感。为了最大限度地减少这种干扰，细胞应该在其天然的多细胞环境中、在尽可能柔和的照明下进行成像。然而，在这些条件下实现详细跟踪三维亚细胞过程所需的时空分辨率具有挑战性：样本引起的像差会降低分辨率和灵敏度，而高分辨率通常需要强烈的激发。刘等人。将非侵入性点阵光片显微镜与像差校正自适应光学相结合，研究体内各种微妙的亚细胞事件，包括有丝分裂期间的细胞器重塑和脊髓发育期间的生长锥动力学。科学，本期第 14 页。 eaaq1392 自适应光学晶格光片显微镜允许在体内自然观察精细的 3D 亚细胞过程。简介 生物体依靠从分子到多细胞的数百万个组件之间复杂、动态、三维 (3D) 的相互作用而生存。要以自然形式可视化这种复杂性，需要在生物体本身的任何空间和时间上以高分辨率成像，因为只有存在调节其生理学的所有环境因素。然而，多细胞系统的光学异质性会导致像差，随着成像深度的增加，这些像差会迅速损害分辨率、信号和对比度。此外，即使没有像差，高分辨率和快速成像通常也伴随着强烈的照明，这会扰乱微妙的亚细胞过程，甚至引入永久性的光毒性效应。 基本原理我们结合了两种成像技术来解决这些问题。第一种是点阵光片显微镜 (LLSM)，它可以快速、重复地将超薄光片扫过感兴趣的体积，同时获取一系列图像，从而构建内部动态的高分辨率 3D 电影。将照明限制在薄平面上可确保体积外部的区域保持不暴露，而从整个平面平行收集荧光则允许使用较低的、扰动较小的强度。第二种技术是自适应光学 (AO)，它测量样品引起的在体积内产生的荧光“引导星”图像的畸变（这种畸变也会影响所获取的光片图像），并通过改变引导星的形状来补偿这些畸变。镜子产生相等但相反的扭曲。结果我们应用 AO-LLSM 在广泛的长度尺度上研究体内的各种 3D 亚细胞过程，从网格蛋白包被的凹坑 (CCP) 的纳米级扩散到发育中的斑马鱼脊髓的 200 μm 轴突引导运动。细胞膜的清晰划分使我们能够在完整生物体的拥挤多细胞环境中通过计算分离和单独研究任何所需的细胞。通过这样做，我们可以比较不同细胞类型的特定过程，例如肌肉纤维和脑细胞中 CCP 内化的速率、发育中的大脑和眼睛的细胞分裂过程中的细胞器重塑，以及免疫细胞和转移性乳腺癌使用的运动机制细胞。尽管大多数例子取自斑马鱼胚胎，但我们还在人类干细胞衍生的类器官、秀丽隐杆线虫和拟南芥叶中证明了 AO-LLSM。 结论 AO-LLSM 对亚细胞过程进行高分辨率活细胞成像，从盖玻片的范围到整个透明生物体内更生理相关的 3D 环境。这为研究不同细胞类型、生物体和发育阶段的细胞内动力学、细胞外通讯和集体细胞行为的表型多样性创造了新的机会。高分辨率体内细胞生物学。 AO-LLSM 允许以高时空分辨率研究其天然多细胞环境中的 3D 亚细胞过程，包括（从左上顺时针方向）脊髓轴突的生长；癌细胞转移；集体细胞运动；内吞作用；微管位移；免疫细胞迁移；和（中心）细胞器动力学。亚细胞动力学的真正生理成像需要研究其母体生物体内的细胞，其中存在驱动基因表达的所有环境线索，因此存在我们实际观察到的表型。完整的理解还需要以高时空分辨率对细胞及其周围环境进行体积成像，而不会对任何一方造成过度的压力。我们将点阵光片显微镜与自适应光学相结合，在大的多细胞体积中实现亚细胞过程的无创无像差成像，包括内吞作用、有丝分裂期间的细胞器重塑以及轴突、免疫细胞和体内转移性癌细胞的迁移。该技术揭示了不同生物体和发育阶段细胞内的表型多样性，并可能为细胞如何利用其内在变异性来适应不同的生理环境提供见解。