【Adv.Mater.】超分辨率超声实现单细胞级示踪，空间精准度突破衍射极限#

Fabrication, physicochemical characterization, and stability of PBCA NB. (A) The Schematic illustration depicts the fabrication of PBCA NB. The schematic was created with BioRender.com. (B) Representative photographs of PBCA NB and MB suspensions after standing for 24 h show that MB forms a floating cake due to buoyancy, whereas NB remains homogeneously dispersed. (C) Coulter counter measurements of MB (red) and NB (blue) samples show a dominant micrometer-scale size distribution for MB centered in the 1–4 µm range, whereas no detectable microscale population is observed for NB. (D) Nanoparticle tracking analysis (NTA) shows the size distribution and concentration of PBCA NB in the nanoscale range. (E) A representative transmission electron microscopy image (TEM, scale bar: 500 nm) shows predominantly spherical, bubble-like PBCA NB, with partial collapse attributed to sample preparation conditions. (F) A representative scanning electron microscopy image (SEM, scale bar: 2 µm) shows spherical, bubble-like PBCA NB. (G) A representative Cryo-SEM image of fractured PBCA NB (scale bar: 500 nm; zoom-in scale bar: 300 nm) reveals distinct internal cavities. (H) DLS measurements of hydrodynamic diameter and PDI over 24 h at 37°C show that PBCA NB remain stable in PBS and in PBS containing 10% FBS (n = 3). Data are shown as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. No statistically significant differences were observed across the evaluated conditions (p > 0.05). (I) DLS size distributions of the NB measured after 24 h of incubation at pHs 7.0 and 4.5 show no significant changes, indicating stability under acidic conditions relevant to intracellular (lysosomal) environments.#

In vitro echogenicity and optimization of cell-labeling with PBCA NB. (A) This schematic illustrates our gelatin phantom used for US imaging, in which a 10% gelatin matrix serves as a US-transparent base, and NB dispersed in 2% gelatin is introduced into a preformed hollow well and solidified by cooling, with degassed water used as the coupling medium. (B) Representative B-mode and contrast-mode US images of gelatin alone and NB-containing gelatin demonstrate detectable acoustic signals from PBCA NB. (C) Schematic illustrating the in vitro NB labeling of J774A.1 macrophages and their embedding in a 2% gelatin phantom for static US imaging. (D) Representative B-mode and contrast-mode US images show cells labeled with NB at different NB-to-cell ratios, with cells labeled at a ratio of 105:1 exhibiting the strongest signal in both imaging modes. (E) Cell viability assessed by XTT assay after NB incubation at varying NB-to-cell ratios shows no significant loss of viability at ratios of 105:1 and below (n = 3). Data are shown as mean ± SD. Statistical significance was determined using a one-way ANOVA followed by Tukey’s post hoc test (**p < 0.01 compared to the 104:1 ratio). (F) Representative US images acquired immediately after NB labeling (0 h) and after 24 h of culture show retained detectability of NB-labeled cells. (G) Quantification of the detected cell number per imaging plane and mean grey value at 0 and 24 h after labeling shows no statistically significant differences (n = 3). Data are shown as mean ± SD. Statistical analysis was performed using an unpaired two-tailed Student’s t-test (ns = not significant, p > 0.05).#

Intracellular localization of NB in cells by confocal microscopy and in vitro cell tracking using ULM. (A) A 3D confocal reconstruction shows intracellular localization of NB-labeled cells, with NB shown in green, cell membranes in red, and nuclei in blue. NB-labeled cells show distinct NB-associated intracellular fluorescence throughout the cytoplasm. (B) A line-scan intensity analysis across a representative cell shows that NB signals are localized between the membrane and nucleus, indicating cytoplasmic internalization. (C) US 3D reconstructions of NB-labeled cells embedded in gelatin at different cell concentrations show concentration-dependent increases in detectable echogenic signals, whereas unlabeled samples show minimal background signal. (D) A schematic illustrates the flow phantom setup used for dynamic US imaging of NB-labeled cells, consisting of a peristaltic pump connected to a perfusable gelatin tunnel. (E) Bright-field microscopy images of unlabeled and NB-labeled macrophages show comparable cell morphology and no apparent aggregation after labeling (scale bar: 50 µm). (F) Representative B-mode and contrast mode US images acquired under flow conditions illustrate enhanced detectability of NB-labeled cells compared with unlabeled cells. (G) ULM-based reconstruction of NB-labeled cell trajectories under flow conditions reveals a laminar flow profile within the channel. The tracks are color-coded by velocity, with the maximum velocity per pixel plotted.#

Preparation and characterization of NB-labeled BMMC. (A) Schematic illustrating the isolation of BMMC from donor mice, including red blood cell lysis, size-based enrichment, centrifugation, and cell counting prior to NB labeling. (B) Time-dependent uptake of rhodamine-labeled NB by BMMC was quantified by fluorescence measurements, showing maximal uptake after 2 h of incubation (n = 3). Data are shown as mean ± SD. (C) Flow cytometry analysis confirms internalization of rhodamine B-labeled NB by viable BMMC following 2 h of incubation. (D) Representative B-mode and contrast mode US images of unlabeled and NB-labeled BMMC embedded in a gelatin phantom demonstrate enhanced echogenicity after NB labeling. (E) Flow cytometry–based immunophenotyping of BMMC before and after NB labeling shows preserved distributions of B cells, T cells, macrophages, dendritic cells, and natural killer cells.#

随后在小鼠乳腺癌模型中，通过腹主动脉插管实施动脉内细胞输注，实时超声成像观察到纳米气泡标记的单核细胞在肿瘤血管内的清晰动态增强信号，而未标记的对照组则完全无法被超声捕获。通过超声局域显微术算法对细胞连续帧信号进行精准定位与追踪，研究团队首次在体内成功重建出高分辨率的细胞运动轨迹图，并将其与微气泡生成的超分辨率血管网络图谱完美融合，实现了对细胞输送血管途径的精确解剖学定位，流式细胞术也同步证实了细胞在肿瘤部位的成功递送。

In vivo tracking of NB-labeled BMMC in murine breast carcinomas using ULM. (A) The experimental timeline and schematic illustrate the experimental workflow of the in vivo cell-tracking, including breast tumor establishment and US imaging steps consisting of baseline acquisition, cell injection, cell infusion recording, application of a destructive pulse, MB injection, MB infusion recording, and post-processing with ULM. (B) Representative contrast mode US images of tumors acquired before and during infusion of unlabeled or NB-labeled BMMC show detectable contrast enhancement only in the NB-labeled group. The green arrows indicate the enhanced signals from NB-labeled cells. The large yellow dashed boxes indicate the specific Regions of Interest (ROIs) selected for subsequent ULM processing displayed in panels F-G. The two rows represent separate acquisitions from different animals, which are marked with a blue vertical line (unlabeled BMMC group) and a green vertical line (NB-labeled BMMC group). (C) Time–intensity curves of tumor contrast signals acquired during infusion of unlabeled BMMC show no appreciable signal enhancement. (D) Time–intensity curves acquired during infusion of NB-labeled BMMC show a significant increase in contrast signals within the tumor. (E) ULM-based reconstruction of in vivo cell tracks shows sparse trajectories in the unlabeled group and robust cell-associated tracks in the NB-labeled group. (F) MIP and corresponding MB track map reconstructed from the MB infusion sequence delineate perfused tumor vasculature within the corresponding ROIs. (G) Merged overlays of the vascular MIP, MB tracks (in red), and cell tracks (in blue) show that NB-labeled cell trajectories predominantly co-localize with perfused vessels. White dashed boxes B, C, and G (merged images) correspond to the zoom-in regions displayed on the right, highlighting the precise co-localization of cell tracks within the microvasculature. (H) Distributions of mean and maximum velocities of per-track extracted from reconstructed cell trajectories are shown for unlabeled and NB-labeled groups. (I) Flow cytometry analysis of single-cell suspensions prepared from excised tumors immediately after imaging confirms the presence of injected cells in both unlabeled and NB-labeled groups.#