【Nat.Methods】400微米活体超分辨！多模态自适应光学显微镜（MOSAIC系统）实现从分子到器官深层活体成像#

a, LLSM image from Supplementary Video 2 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 1,000 × 750 × 10 µm3 volume. Scale bar, 50 µm. b, Zoomed-in views of cell division events from a. Magenta panel (left), volume rendering of a metaphase cell having a large ER protrusion; blue panel (middle), four points in nominal cell division from metaphase to telophase; yellow panel (right), six points during a rare tripolar mitotic event. Scale bar, 10 µm. c, Label-free OI imaging at 1 Hz (Supplementary Video 3) 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 1,218 × 975 µm2 field of live U2OS cells at 1 Hz. Scale bar, 200 µm. Right, zoomed-in view (white box) of a single dividing U2OS cell at four points, showing condensed chromosomes (arrows). Scale bar, 10 µm (right).#

a, Nuclear imaging (SPY 505) and single-particle tracking with LLSM, visualizing molecular trajectories and diffusion dynamics of SOX2 (HaloTag-PA-JF646) in mouse embryonic stem (mES) cells at 50 Hz. Scale bar (left), 5 µm. Scale bar (top right), 2 µm. The probability density histogram of log10 diffusion coefficients is binned from −3 to +3 in 0.1-unit increments. b, Top: LLS-SIM xy MIP of hTERT-RPE1 cells expressing ER (StayGold-ER, gray) and Golgi (β4Gal-HaloTag9 labeled with JFX549, orange) markers, from Supplementary Video 6. 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, Timelapse 3D-SIM (Supplementary Video 7) 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 Supplementary Video 8. Scale bar, 10 µm. f, Timelapse sequence of the correlative imaging shown in e. Scale bar, 10 µm.#

a, Two-color 3D DNA-PAINT of the mitochondrial marker TOMM20 and the nuclear envelope protein marker Lamin A/C in U2OS cells. Left to right, overview MIP of a 180 × 200 × 17 µm3 FOV (scale bar, 20 µm), zoomed-in 3D rendering of a single cell (boxed region, 28 × 23 × 5.4 µm3), its xz orthoslice (scale bar, 2 µm) and the close-up of nuclear invaginations (boxed region, 4.7 × 4.5 × 5.4 µm3). See also Supplementary Video 9. b, ExLLSM MIP overview of a 2,000 × 2,375 × 98 µm3 human hippocampal tissue section from a patient with AD after 4× 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-in 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.#

a, Comparison of LLSM imaging in a zebrafish xenograft system (55 × 183 × 50 µm3) showing actin-labeled MDA-MB-231 human breast cancer cells (magenta) within the zebrafish vasculature (green), both without AO (left) and with AO correction plus deconvolution (right). Insets, corresponding Fourier spectra (at gamma = 0.3) of the magenta channel. Scale bar, 10 µm. b, Timelapse imaging of a capturing cancer cell dynamics and vascular damage during extravasation. Scale bar, 20 µm. c, Zebrafish tail fin volume (216 × 272 × 37 µm3) at 66 h 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 (Supplementary Video 11), 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 DNA Helicase B (DHB), in segmented and computationally separated cells (216 × 173 × 37 µm3 pre-separation, inset). Peripheral cells have the highest fraction in G2 (Supplementary Video 12).#

在活体多细胞生物成像中，自适应光学技术展现出核心优势。通过对斑马鱼胚胎进行多区域拼接校正，系统完美恢复了空间频率，清晰记录了人类乳腺癌细胞在血管中的游走、外渗及血管损伤。在活体小鼠大脑功能显微成像实验中，系统成功对抗了颅窗和脑组织像差，在皮层400微米深处清晰解析出单个树突棘结构，并将钙信号信噪比显著提升，捕获到比未校正状态多出2.5倍的树突棘钙瞬变事件，全面证实了其活体研究价值。

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 × 57 × 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, Fourier spectrum of reconstructed mitochondria (Supplementary Video 14). c, AO-LLS-SIM MIPs of orthogonal slabs (3-µm thick) in the hindbrain from a 56 × 56 × 40 µm3 volume (inset) in a 14 hpf zebrafish with mitochondria in RGB colors and membrane in gray. 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 × 319 × 84 µm3 image volume spanning brain, muscle and notochord in a membrane-labeled 7 dpf zebrafish. Scale bar, 50 µm. Bottom, zoomed-in views comparing muscle and neural progenitor cells imaged by ISM with and without AO (Supplementary Video 16). g, AO-ISM MIP (354 × 332 × 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, Timelapse AO-ISM showing xy orthoslices of a migrating cell in the neuromast. Scale bar, 10 µm.#