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

Abstract

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.

Fig. 1. Adaptive optical lattice light-sheet microscopy (AO-LLSM)

(A) Simplified microscope schematic (fig. S1 shows a detailed version). EO, excitation objective; DO, detection objective; SH, sample holder. (B) xy and xz maximum intensity projections (MIPs) of the point spread function (PSF; top two rows) and corresponding optical transfer function (OTF; bottom two rows) of the microscope under five different degrees of AO correction (columns), as measured from a 200-nm fluorescent bead in an aberrating agarose gel. Insets show the corrective wavefronts applied. Arrowheads indicate lateral and axial aberrations. Scale bar, 1 μm. (C) MIPs and corresponding OTFs of the uncorrected (left column) and fully corrected (right column) bead images from (B), after deconvolution using the aberration-free reference PSF. Scale bar, 1 μm. (D) Cellular trans-Golgi, mitochondria, and plasma membranes in the spine of a live zebrafish embryo 24 hpf. Shown are unprocessed data without AO correction (left column), deconvolved data without AO correction (center), and deconvolved data after AO correction (top and right) (movie S2). MIP views (bottom two rows) of the Fourier transform (FT) of the data in all three cases indicate their respective degrees of information recovery. (E) Four different levels of correction, shown for orthoslices of a live human stem cell–derived organoid grown in Matrigel and gene-edited to express endogenous levels of clathrin and dynamin in coated endocytic pits (Movie 1). Scale bar, 5 μm.

Fig. 2. Clathrin dynamics in zebrafish

(A) Computationally separated muscle fibers (e.g., green arrowheads) and vascular endothelial cells (e.g., magenta arrowheads), both expressing DsRed-CLTA to highlight CCPs and CCVs, from muscle tissue in a 75-μm by 99-μm by 41-μm region (upper inset) of the tail of a developing zebrafish larva 80 hpf (Movie 2). Brighter clathrin puncta were observed in the endothelial cells (lower inset). Scale bars, 10 μm. (B) Computationally separated muscle fibers from a region (lower inset) in the tail of a zebrafish embryo 50 hpf coexpressing an mCardinal-PM marker (red) and mNeonGreen-CLTA (green). Individual CCPs and CCVs and larger clathrin-rich vesicles (arrowheads) are visible (Movie 3). Scale bars, 10 μm. (C) Spatial distribution and dynamics of CCPs and CCVs tracked for 12 min at 7.5-s intervals in one muscle fiber from (B), showing CCPs localized at t-tubules (top left) and diffusion and lifetime characteristics for CCPs and CCVs across the cell (top right). A MIP through a 2-μm-thick slab at three consecutive time points (bottom) shows examples of a pinned CCV (arrowheads 2) and slowly diffusing (arrowheads 1) or rapidly shuttling CCVs (arrowheads 3) (movie S3). Scale bar, 10 μm. (D) Effect of AO on the measured quantity, intensity, and localization precision of CCPs and CCVs in the organoid in Fig. 1E and the zebrafish in (B). (E) Comparative distribution of CCP and CCV lifetimes (left) and intensity cohorts grouped by their lifetimes (right) in the brain and muscle of a developing zebrafish embryo 55 hpf.

Fig. 3. Organelle morphologies and dynamics in zebrafish

(A) Computationally separated neural progenitor cells from a 70-μm by 35-μm by 35-μm region (inset) in the brain of a developing zebrafish embryo expressing GalT-mNeonGreen, tagRFP-Sec61β, and Citrine as markers of the trans-Golgi, ER, and PM, respectively, with additional labeling by MitoTracker Deep Red dye (Movie 4). Scale bars, 10 μm. (B) Changing morphologies of the organelles in the specific cell outlined in (A) at three time points through mitosis. Arrowheads indicate mitotic blebs. Scale bar, 10 μm. (C) MIP views from 1-μm-thick orthogonal slabs within the eye of a zebrafish embryo 30 hpf, showing PM (blue) and endomembranes (orange) (Movie 5). Scale bars, 10 μm. (D) Six time points from Movie 5, showing PM blebs (white arrowheads) during mitosis and the exclusion of endomembranes in early blebs (green arrowheads). (E) Correlation between nuclear volume and total cell volume in the eye and ear (Pearson’s coefficient, 0.9 and 0.8, respectively). (F) Different morphologies of trans-Golgi (top) near the spine of a zebrafish embryo 24 hpf and distribution of trans-Golgi volume in different cell types and at different developmental stages (bottom).

Fig. 4. AO-LLSM over large volumes

(A) Aberration-corrected volume rendering over 213 μm by 213 μm by 113 μm in the tail region of a live zebrafish embryo 96 hpf expressing PM-targeted EGFP, assembled from independently corrected subvolumes of 7 by 7 by 3 tiles (movie S4). (B) Increasing effectiveness of correction, as seen in orthogonal MIPs from 3-μm-thick slabs, under different scenarios: no AO (left column), AO correction from the center tile applied globally (middle column), and independent AO correction in each tile (right column) (fig. S11). Insets compare, at higher magnification, the quality of correction at the center tile (orange boxes) versus at the tiles at the periphery of the tail (blue boxes). Tile boundaries are shown in white. Scale bar, 30 μm. (C) A 5 by 4 by 7 set of measured excitation (left column, top) and detection (left column, bottom) aberrations which, after AO correction, yields diffraction-limited imaging over a 170-μm by 185-μm by 135-μm volume (left column, center) in the spine of a zebrafish embryo 30 hpf (Movie 6). Red and green arrowheads indicate excitation aberrations in specific tiles before and after passage through the notocord, respectively. The yellow arrowhead indicates a tile with a large detection aberration deep within the specimen. Orthoslices before (middle column) and after (right column) AO correction show increased aberration but continued recovery of high resolution at progressively greater depth. Scale bar, 30 μm. (D) Aberration-corrected volume renderings over 156 μm by 220 μm by 162 μm in the spine of a zebrafish embryo, at three points from a time series at 30-min intervals (movie S5), flanked by excitation and detection path aberrations at those points. Those tiles whose corrections were updated at a given time point are marked in green.

Fig. 5. Organelle diversity across the zebrafish eye

(A) Tiled array used to provide AO correction across the eye of a developing zebrafish embryo 24 hpf (Movie 7). Scale bar, 30 μm. (B and C) Distribution of three different types of organelles across the volume assembled from the tiles in (A). Scale bars, 30 μm. (D) Computationally separated cells across the eye, with the organelles colored as indicated. Scale bar, 30 μm. (E) Organelle morphologies in cells of three different types within the eye. Scale bar, 30 μm. (F) Orthoslices at six different time points highlighting cell divisions (white and green arrowheads, left panel) at the apical surface of the retinal neuro-epithelium and mitochondria (orange arrowheads) present from the apical to the basal surface in one dividing cell. Scale bar, 30 μm.

Fig. 6. 3D cell migration in vivo

(A) Two views of newly differentiated neurons highlighted by Autobow labeling in a 60-μm by 224-μm by 180-μm section of the spinal cord of a zebrafish embryo 58 hpf (Movie 8). Magenta and yellow arrowheads show neurons differentiated before and after Autobow expression, respectively. (B) Increase in the density of rostro-caudally projecting axons over time. Scale bar, 20 μm. (C) Sagittal (top) and transverse (bottom) views of the growth cones of four rostrocaudally projecting axons. Scale bars, 10 μm. (D) Sagittal (top) and transverse (bottom) views of the growth cones of three dorsoventrally projecting axons. Scale bars, 10 μm. (E) Time-coded color overlay of an immune cell migrating within the perilymphatic space next to the inner ear of a live transgenic zebrafish embryo 70 hpf expressing PM-targeted Citrine (Movie 9 and fig. S12). Texas Red dextran particles are shown in blue. Scale bar, 10 μm. (F) Changing morphologies of two different immune cells (top and bottom rows), one showing internalized dextran particles (blue) (fig. S13). Scale bar, 5 μm. (G) MDA-MB-231 human breast cancer cell (green) rolling in a blood vessel (magenta) in a zebrafish embryo 48 hpf. (H) Another MDA-MB-231 cell crawling through a blood vessel. (I) A partially extravasated MDA-MB-231 cell, showing an increasingly complex morphology over time (Movie 10 and fig. S1). Scale bars, 10 μm in (G) to (I).

Movie 1. Endocytosis in a human stem cell–derived organoid

Gene-edited clathrin (magenta) and dynamin (green) before and after adaptive optical correction and deconvolution, showing comparative xy and xz orthoslices, volume renderings, and postcorrection tracking of the motion and lifetimes of individual CCPs and CCVs over 120 time points at 1.86-s intervals (Fig. 1E and fig. S5).

Movie 2. Clathrin-mediated endocytosis in vivo

Dynamics of CCPs and CCVs over 15 min at 10-s intervals in the dorsal tail region of a zebrafish embryo 80 hpf. Segmented cells reveal brighter clathrin puncta at the vascular endothelium than at muscle fibers (Fig. 2A).

Movie 3. Clathrin localization in muscle fibers

PM (red) and clathrin (green) in the tail of a zebrafish embryo 50 to 55 hpf, showing xy and xz orthoslices before and after AO correction and deconvolution, dynamics of individual CCPs and CCVs at and between t-tubules, large clathrin clusters and small clathrin puncta in volume-rendered and segmented cells, and tracked CCPs and CCVs in a segmented cell (Fig. 2, B to E; fig. S8; and movie S3).

Movie 4. Subcellular imaging of organelle dynamics in the early zebrafish brain

Dynamics of PM (green or gray) and trans-Golgi (green), ER (magenta or red), and mitochondria (cyan) within neural progenitor cells over 200 time points at 44-s intervals from 14.0 hpf, showing complexity within the tissue, cross-sectional slab views through cells, sequential division of adjacent cells, segmentation and separation of all cells, and morphological changes to organelles during mitosis in one such cell (Fig. 3, A and B, and figs. S9 and S10).

Movie 5. Membrane dynamics in the zebrafish eye

PM (blue) and the endomembrane system (orange) 30 hpf viewed as xy orthoslices, cell divisions in a 1-μm-thick slab, and volume-rendered PM dynamics across the eye at 43.8-s intervals for 200 time points (Fig. 3, C to E).

Movie 6. Tiled AO correction for imaging large volumes

A 170-μm by 185-μm by 135-μm volume from the dorsal surface to the notochord in a PM-labeled zebrafish embryo, showing increasing aberration but continued full correction at increasing depth; corrective excitation and detection wavefronts in each of the tiled isoplanatic subvolumes of 5 by 4 by 7 tiles; and four views of PM dynamics within the complete volume from 30 to 39.5 hpf, imaged at 7.5 min intervals (Fig. 4C).

Movie 7. Organelle dynamics across the zebrafish eye

PM (cyan), trans-Golgi (green), ER (magenta), and mitochondria (brown) across a 128-μm by 150-μm by 75-μm volume assembled from subvolumes of 4 by 4 by 3 tiles, showing orthoslices in a single tile, volume-rendered tiles before assembly into the combined volume, organelles in the combined volume, dynamics over 30 time points from 24.0 to 26.8 hpf in a 1-μm-thick slab through the combined volume, dynamics in perpendicular orthoslices, and organelle morphologies in different cell types in the computationally expanded volume (Fig. 5).

Movie 8. In vivo imaging of spinal cord neural circuit development

Autobow-labeled, newly differentiated neurons expressing stochastic combinations of three fluorophores in a zebrafish embryo, showing corrective excitation and detection wavefronts in subvolumes of 5 by 2 by 1 tiles, with scrolling updates at one tile (green box) per time point; AO-corrected orthoslices and volume-rendered views in each color channel 58 hpf; and axon pathfinding in each color channel from 58 to 70 hpf (Fig. 6, A to D; fig. S12; and movie S6).