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

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.