A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Abstract

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

Fig. 1.. Large field-of-view dynamic imaging with 3D lattice light-sheet or 2D label-free oblique illumination microscopy.

(a) LLSM image from Movie S2 of LLC-PK1 cells expressing Calnexin-mEmerald (ER) and H2B-mCherry (nuclei). Maximum-intensity-projections (MIPs) show xy (top) and xz (bottom) views of the 1000 x 750 x 10 μm³ volume. Scale bar, 50 μm. (b) Zoomed views of cell division events from (a). Left: volume rendering of a metaphase cell having a large ER protrusion; Middle: four points in nominal cell division from metaphase to telophase; Right: six points during a rare tripolar mitotic event. Scale bar, 10 μm. (c) Label-free OI imaging at 100 Hz (Movie S3) captures HeLa cell lamellipodial ruffling and replication of contaminating bacteria. Scale bar, 10 μm. Right: Magnified view (magenta box) of three bacterial division events (arrows). Scale bar, 2 μm. (d) Tiled label-free imaging over a 1218 x 975 μm² field of live U2OS cells at 1 Hz. Scale bar, 200 μm. Right: Zoomed view (white box) of a single dividing U2OS cell at four points, showing condensed chromosomes (arrows). Scale bar, 10 μm (right).

Fig. 2.. Multimodal super-resolution imaging of subcellular dynamics.

(a) ) Nuclear imaging (SPY 505) and single-particle tracking with LLSM, visualizing molecular trajectories and diffusion dynamics of SOX2 (HaloTag-PA-JF646) in mESC cells at 50 Hz. Scale bar (left panel), 5 μm. Scale bar (upper right panel), 2 μm. (b) Top: LLS-SIM xy MIP of hTERT-RPE1 cells expressing ER (StayGold-ER, gray) and Golgi (β4Gal-HaloTag9 labeled with JFX 549, orange), from Movie S6. Insets: corresponding xy and yz OTFs. Scale bar, 25 μm. Bottom: Volume renderings from boxed regions above showing ER and Golgi organization. Scale bar, 2 μm. (c) Comparison of widefield and 3D-SIM imaging in hTERT-RPE1 cells with mitochondria (COX8a-StayGold, cyan) and Golgi marker (orange). Scale bar, 25 μm. Insets: 3D-SIM OTFs. (d) Time-lapse 3D-SIM (Movie S7) captures mitochondrial and Golgi dynamics during cell division (white box from (c)). Scale bar, 5 μm. (e) Correlative optical microscopy applying multiple imaging modalities (widefield, 3D-SIM, OI, LLS, and LLS-SIM) to a dividing hTERT-RPE1 cell with labeled mitochondria (blue-green) and Golgi (orange), from Movie S8. Scale bar, 10 μm (f) Time-lapse sequence of the correlative imaging shown in (e). Scale bar, 10 μm.

Fig. 3.. Volumetric imaging with nanoscale resolution.

(a) Two-color 3D DNA PAINT of the mitochondrial marker TOMM-20 and the nuclear envelope protein marker Lamin A/C in U2OS cells. Left to right: Overview MIP of a 180 x 200 x 17 μm³ FOV (scale bar, 20 μm), zoomed 3D rendering of a single cell (boxed region, 28 x 23 x 5.4 μm³), its xz orthoslice (scale bar, 2 μm), and the close-up of nuclear invaginations (boxed region, 4.7 x 4.5 x 5.4 μm³). See also Movie S9. (b) ExLLSM MIP overview of a 2,000 x 2,375 x 98 μm³ human hippocampal tissue section from an Alzheimer disease patient, after 4x expansion. NF-200 (blue) and MBP (yellow) label neurofilaments and myelin sheaths, respectively. Scale bar, 500 μm. (c) Zoomed view from (b) showing neurofilament and myelin sheath ballooning. Scale bar, 20 μm. (d) Nanoscale structure of axon and myelin sheath blebs. Scale bar, 2 μm. (e) Zoomed region from (b) highlighting clustering of NF-200 protein. Scale bar, 20 μm. (f) Nanoscale structure of an individual NF-200 cluster. Scale bar, 2 μm. Scale bars throughout represent pre-expansion dimensions.

Fig. 4.. Observing cellular and subcellular dynamics within zebrafish embryos.

(a) Comparison of LLSM imaging in a zebrafish xenograft system (55 x 183 x 50 μm³) showing actin-labeled MDA-MD-231 human breast cancer cells (magenta) within the zebrafish vasculature (green), both without AO (left) and with AO correction plus deconvolution (right). Insets: corresponding FFTs (at gamma = 0.3) of the magenta channel. Scale bar, 10 μm. (b) Time-lapse imaging of (a) captures cancer cell dynamics and vascular damage during extravasation. Scale bar, 20 μm. (c) Zebrafish tail fin volume (216 x 272 x 37 μm³) 66 hours post-amputation showing plasma membranes and nuclear histones. Scale bar, 50 μm. (d) Cellular and subcellular events during the initial stages of regeneration after amputation (Movie S11), showing: extracellular vesicle release from a cell adjacent to the cut site (yellow box); anchoring fibril dynamics in the epidermal basement membrane (red box); a mesenchymal cell fusion event (gray box); and a transiently trapped red blood cell during remodeling of the caudal vascular plexus (green box). Scale bar, 5 μm. (e) Visualization of cell cycle state across the fin, based on the cytoplasmic to nuclear fluorescence ratio of CDK biosensor DHB, in segmented and computationally separated cells (216 x 173 x 37 μm³ pre-separation, inset). Peripheral cells have the highest fraction in G2 (Movie S12).

Fig. 5.. Super-resolution imaging in vivo.

(a) MIPs of raw zebrafish membrane images with LLS-SIM illumination without (top) and with AO (bottom). Ticks show the expected positions of the LLS pattern excitation maxima. Scale bar, 5 μm. (b) Orthoslices (180 nm thick) in xy, xz, and yz from an AO-LLS-SIM reconstruction (61 x 57 x 40 μm³) in the eye of a 14 hpf zebrafish embryo expressing mitochondrial (magenta) and plasma membrane (cyan) markers. Scale bar, 10 μm. Top left inset: FFT of reconstructed mitochondria (Movie S14). (c) AO-LLS-SIM MIPs of orthogonal slabs (3 μm thick) in the hindbrain from a 56 x 56 x 40 μm³ volume (inset) in a 14 hpf zebrafish with mitochondria in RGB colors and membrane in grey. Mitochondria in each cell are color-coded by the ratio of total mitochondrial length to cell volume. Scale bar, 10 μm. (d) Cutaway view of segmented mitochondria in four different segmented cells. Scale bar, 4 μm. (e) Cutaway view of one cell from the volume, showing mitochondrial rearrangements to the daughter cells during division. Scale bar, 4 μm. (f) Top: ISM MIP views before and after AO correction of a 0.324 μm xy orthogonal slab within a larger 336 x 319 x 84 μm³ image volume spanning brain, muscle, and notochord in a membrane-labeled 7 dpf zebrafish. Scale bar, 50 μm. Bottom: Zoomed views comparing muscle and neural progenitor cells imaged by ISM with and without AO (Movie S16). (g) AO-ISM MIP (354 x 332 x 16.3 μm³) of a dorsal-mounted, membrane labeled, 7 dpf zebrafish. Scale bar, 50 μm. (h) Volume rendering with xy and yz orthoslices through a neuromast. Scale bar, 10 μm. (i) Time-lapse AO-ISM showing xy orthoslices of a migrating cell in the neuromast. Scale bar, 10 μm.

Fig. 6.. In vivo AO two-photon structural and functional imaging in the mouse cortex.

(a, b) xy (a) and xz (b) MIPS of a 100x100x400 μm³ volume starting at a depth of 60 μm in a Thy1-YFP-H mouse after AO correction and deconvolution (Movie S17). Wavefronts at right in (b) show the measured aberration at 25 μm intervals from 60 to 260 μm deep. Scale bar, 10 μm. (c) Zoomed xy MIPS from four color coded subvolumes as shown in (a) and (b), before AO and after AO plus deconvolution (d) Functional imaging of neural activity 40 μm deep in the cortex of a GCaMP7s mouse (Movie S18). Top: color-coded time projection. Scale bar, 20 μm. Middle: Single activated dendrite from the boxed region at top, before and after AO. Bottom: Comparison of raw spikes from the indicated dendritic spine acquired sequentially without and with AO. (e) Left: Traces comparing measured calcium transients from the spines indicated in (d). Right: Distribution showing that AO correction yielded a ~2.7x more detectable dendritic spine calcium spikes than without AO over the same duration. (f) Two-color in vivo AO-TPM of dendrites and vasculature, the latter labeled by injection of TexasRed, over a 500 x 500 x 100 μm³ FOV (Movie S19) with 5 x 5 x 1 AO corrective tiles. Scale bar, 50 μm. (g) Left: Zoomed view of the boxed region in (f). Scale bar, 10 μm. Right: individual dendritic spines. Scale bar, 2 μm. (h) Additional spines in a further magnified view of the boxed region in (g). Scale bar, 5 μm.