Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore

Abstract

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Extended Data Figure 1.. Rhobo6 cell impermeability and additional photophysical characterization

a, Comparison between Rhobo and Rhobo6 cell permeability over time. PC3 cells were incubated with Rhobo or Rhobo6 at 5 μM concentration in serum-containing media. Wells were then imaged directly after addition of dye (t = 0 h) and following a 1, 2, and 6 h incubation at 37 °C and 5% CO₂. Cell surface signal is absent in this experiment due to the presence of serum-containing media (cf. Extended Data Fig. 3d) and excitation at 488 nm (cf. (e)-(h)). b, Quantified intracellular signal for Rhobo and Rhobo6 over time as determined by manually drawn regions of interest of N = 9 cells per condition. Error bars represent SEM. c, Monosaccharides and monosaccharide analogs used in (d). d, Quantified fluorescence of Rhobo6 measured by exciting at 555 nm and detecting fluorescence intensity maxima between 570 and 630 nm. All sugars were prepared in PBS solutions at 200 mM with 5 μM Rhobo6, pH 7.3-7.4, and incubated for 1 h at room temperature. N = 3, error bars represent SEM. P values were determined by unpaired t-test with Welch’s correction, relative to dye only control; *P < 0.05. e, Relative change in extinction coefficient between bound and unbound Rhobo6 as function of excitation wavelength calculated as (ε_bound-ε_unbound)/ε_unbound. Position of standard laser lines 488 nm and 561 nm are shown to highlight that longer wavelength excitation preferentially excites bound dye, enhancing observed contrast. f, Emission spectra of bound and unbound Rhobo6 normalized to 488 nm excitation (cf. Methods). A standard emission filter for green fluorescence (545 nm short pass [SP]) will preferentially collect light from unbound dye, reducing observed contrast. g, Emission spectra of bound and unbound Rhobo6 normalized for 561 nm excitation. A standard emission filter for red fluorescence (575 nm long pass [LP]) will preferentially collect light from bound dye, enhancing observed contrast. h, Measured contrast (calculated as ΔF/F between bound and unbound Rhobo6), as function of emission wavelength for 490 nm and 561 nm excitation. The plot agrees with renormalized spectral data in (f)-(g), with measured optimal contrast at 561 nm excitation and 570-580 nm emission. i, Two-photon excitation spectra for Rhobo6 in 100 mM phosphate buffer, pH 7.4 (unbound state) and 100 mM phosphate buffer containing 1 M galactose, pH 7.4 (bound state). The measured excitation spectrum of Rhodamine B is shown for reference.

Extended Data Figure 2.. Application of Rhobo6 to a commercial glycan array

a, Normalized fluorescence signal of Rhobo6 measured by quantifying three glycan arrays with four glycan replicates each, printed on a single glass slide. Fluorescence signal was background corrected and normalized within each array. N = 12 for glycans, N = 24 for negative control, error bars represent standard deviation. Statistical significance was determined through Dunnett-corrected t-test for multiple comparisons to a negative control group, with assumption of unequal variance across groups; NS = not significant (orange text), ***P < 0.0005. For all glycans not marked “NS”, average signal was confirmed to be greater than two times the standard deviation of local background. b, Array glycans and glycan linkers. Glycans marked “NS” in (a) are colored orange. Note that linker “Sp4” carries a carboxylic acid, which is negatively charged at physiological pH.

Extended Data Figure 3.. In vitro and in cellulo Rhobo6 characterization, related to Fig. 2.

a, Representative field of views from triplicates shown in Fig. 2b. Contrast is normalized within each sample type (columns). Signal intensity is reduced upon treatment, as quantified in Fig. 2c. b, Frames extracted from 9.6 h imaging, at one frame per minute, on a coated aggrecan substrate incubated with 5 μM Rhobo6. ROIs analyzed in (c) are highlighted in the first frame. c, Time trace of signal, calculated as mean intensity within three different manually traced ROIs, from (b). Photobleaching was not observed over the course of imaging. d, Effect of imaging buffer, mucin expression, and cell fixation on cell surface Rhobo6 signal. MCF10A+GFP-MUC1ΔCT were incubated with doxycycline to induce GFP-tagged mucin expression, and imaged in different buffers containing 5 μM Rhobo6. PBS shows the best signal to background ratio. Cell surface signal can be partially competed upon addition of 200 mM sorbitol (cf. Extended Data Figure 1d), confirming that Rhobo6 binding is diol-dependent and reversible. DMEM/F12 media contains 17.5 mM d-Glucose, likely contributing to a higher background than PBS. Supplementation of DMEM/F12 with the remaining complete media components (cf. Methods) results in the lowest observed signal-to-background ratio, likely due to the abundance of glyconjugates in serum. MCF10A cells that were not induced to express GFP-MUC1ΔCT were also imaged in PBS and exhibited dramatically reduced cell surface Rhobo6 labeling, possibly due to the low density of binding sites on these cells. Finally, mucin-expressing cells were imaged after fixation with 4% paraformaldehyde; as a result of the fixation, membrane integrity is compromised, allowing Rhobo6 to accumulate in the cytosol. Rhobo6 is therefore not suitable for use with chemically fixed samples as it will accumulate non-specifically in fixed cells.

Extended Data Figure 4.. Rhobo6 toxicity, reversibility in comparison to Rhobo, related to Fig. 3

a, Brightfield images of a representative pair of glands from Fig. 3b. b, Raw image of embryonic salivary gland labeled with Rhobo6 reported in Fig. 3c. Inset shows comparison between raw and denoised dataset, confirming no visual artifacts are introduced in the process. Denoise was performed through Nikon NIS Elements AI Denoise (cf. Supplementary Table 1). Line plot of red region is reported in (c). c, Line plot of red outlined region from (b). Denoising increases signal to noise without altering biological features. d, Mouse embryonic salivary glands labeled with 5 μM Rhobo or Rhobo6, along with Hoechst (nuclear stain) and NucSpot650 (dead cell stain). Glands were incubated for 1 h with all probes and imaged. Rhobo6 labeling is confined in the extracellular space, but colocalizes with dead cells due to compromised membrane integrity (arrows). On the other hand, Rhobo labeling accumulates intracellularly in both live and dead cells. e, Glands from (b) were washed three times over the course of 3 h with DMEM/F12. Rhobo6 labeling was rapidly reversible, while Rhobo labeling was not diminished by washing over the course of the experiment. Rhobo images are contrast normalized across (d) and (e) and Rhobo6 images are independently normalized across (d) and (e). Notably, Rhobo is not able to label structures of the extracellular matrix in these glands, likely due to cytosolic sequestration. f, Representative STED image of mouse pancreatic ECM obtained by imaging freshly excised tissue labeled with Rhobo6. Depletion was achieved with a 660 nm laser. g, Three regions of interest (ROIs) from (f), comparing confocal and STED imaging. Contrast is not normalized across imaging conditions and fields of view. Images were denoised (cf. Supplementary Table 1). h, Intensity plot of red line from (g) displaying the increased resolving power achieved by STED microscopy when compared to diffraction-limited confocal microscopy.

Extended Data Figure 5.. ECM labeling in non-mammalian organisms.

a, Volume of an adult Drosophila brain labeled by bathing with 5 μM Rhobo6 in saline. Imaging at different planes reveals labeling surrounding landmarks in the fly brain, including neuron tracts between the optic lobe and the central brain, the central complex, and the mushroom body. Images were denoised (cf. Supplementary Table 1). b, Mean intensity projection of a confocal volume capturing the mushroom body in a Drosophila brain. The sample endogenously expressed GFP in neuronal cells and was labeled with Rhobo6 upon dissection (cf. Methods). Images were denoised (cf. Supplementary Table 1). c, Brightfield and confocal fluorescence images of whole C. elegans injected with 10 pL of 100 μM Rhobo6 in PBS containing 1% DMSO. Contrast is normalized between PBS-injected and Rhobo6 injected animals. Inset is a crop and enlargement of the oviduct region. d, Max intensity projections of confocal volumes taken at the oviduct of animals endogenously expressing Nidogen-1-mNeonGreen (Nid-1-GFP) to highlight oviduct surfaces, co-localized to Rhobo6 labeling. Rhobo6 signal is enriched at the sp-ut valve within the lumen of the oviduct. Rhobo6 does not label the Nid-1-rich oviduct basement membrane, which is not in contact with the lumen of gonad arm. e, Time course of wound healing in zebrafish larvae (8 d.p.f.) incubated with 5 μM Rhobo6 in tank water. Tail nicks (arrows) were necessary for dye delivery. Structures of the tail ECM and notochord are labeled. f, Max intensity projection of an Arabidopsis root after labeling overnight with 5 μM Rhobo6 in pure water. Root cell surfaces are labeled.

Extended Data Figure 6.. Comparison between 2-photon Rhobo6 imaging and label-free ECM imaging techniques

a-c, Gut (jejunum), muscle (masseter), and pancreas from both a control mouse and a mouse retroorbitally injected with 100 nmol of Rhobo6 were imaged using second harmonic generation microscopy (SHG), two photon excitation autofluorescence (2P autofluorescence, also known as TPEF) and two photon excitation fluorescence (2P Rhobo6). Fields of view were imaged with a low light dosage highlighting the efficiency of 2P Rhobo6 compared to SHG and TPEF, followed by a high light dosage for SHG and TPEF, in order to obtain contrast with those methods (cf. Supplementary Table 1). Contrast was normalized across all low dosage images within each tissue type. High dosage SHG images are normalized to each other and high dosage TPEF images are not normalized to any other image. Reported laser power is average measured power at sample plane for 120 fs pulse at 80 MHz repetition rate.

Extended Data Figure 7.. Immunostaining of wild-type and tumor-bearing mouse mammary glands

Immunofluorescence of fixed and wholemounted mammary glands using phalloidin (yellow) and CNA35-GFP (cyan) to mark filamentous actin and fibrillar collagen, respectively, and DAPI to stain cell nuclei (magenta). Two fields of view are shown for each of the mammary glands presented in Fig. 5, resected from the wild-type mouse (left) and MMTV-PyMT mouse 1.

Extended Data Figure 8.

Chemical characterization of Rhobo6. a, Analytical liquid chromatography trace with absorbance detection at 254 nm (top) and mass spectrum of main peak (bottom). b, Stability of Rhobo6 at room temperature in 1:1 DMSO:PBS over time, as assessed by HPLC with absorbance detection at 254 nm. Calculated Rhobo6 purity ranged from 95-96% over the 72 h period.

Figure 1.. Photophysical characterization of the glycan-binding fluorophore Rhobo6.

a, ECM labeling strategy. A cell impermeable dye is added to a biological sample such that it disperses into extracellular spaces. Upon reversible association with glycoconjugates (colored shapes) of the extracellular matrix, the dye increases its fluorescence output. b, Rhobo6 structure and propensity for glycan binding. The carboxylic acid on the 6-position of Rhobo6 (red numbering) is charged at physiological pH, rendering the molecule cell impermeable. The pK_a for ortho-aminomethylphenyl boronic acid is within the range of 5 to 7, meaning the boronate and borate ester dominate in aqueous buffer at physiological pH. Rhobo6 is therefore expected to carry a net charge of negative three. c, Absorption and normalized emission spectra for Rhobo6 in unbound (5 μM dye in PBS) and bound (5 μM dye in PBS containing 2 M sorbitol) states. Emission spectra were measured with excitation wavelength at 490 nm. For 2P spectra see Extended Data Fig. 1i. d, Table of photophysical properties. Molar extinction (ε) is reported at peak absorption. Quantum yield (Φ) is measured as average value measured between 475 nm and 535 nm. Contrast is measured as relative fluorescence signal change between bound and unbound states (ΔF/F), when exciting at 561 nm and detecting fluorescence signal at 575 nm. Because of redshift in both absorption and emission, this value is highly dependent on both excitation and emission parameters (see also Extended Data Fig. 1e–h).

Figure 2.. In vitro and in cellulo validation of Rhobo6 labeling.

a, Rhobo6 labeling of purified ECM components. Substrates were prepared as glass coatings or gels (cf. Methods), and incubated with Rhobo6 at 5 μM for 1 h in PBS. Images were acquired with a confocal microscope. Contrast is not normalized across images. b, ECM components were treated with 10 mM sodium periodate (blue) or with Chondroitinase ABC (green), and signal intensity quantified from confocal microscopy images. For representative images used for quantification, see Extended Data Figure 3a. N = 3, error bars represent SEM. P values were determined by using two-tailed t-test; **P < 0.005; ***P < 0.0005. c, Spectral imaging at the boundary of a collagen I gel and the surrounding buffer containing 5 μM Rhobo6, performed via excitation scan 500-566 nm and detection of fluorescence at 575-630 nm (cf. Supplementary Table 1). Intensity contrast image (left) obtained at 560 nm excitation with manually traced ROIs to capture an area rich in collagen fibers and an area within the surrounding buffer. Excitation spectra (right) corresponding to the manually drawn ROIs. d, A spectral contrast image generated by plotting excitation maxima for each pixel in (c). Binning = 2 pixels for the image shown. e, Time course of Rhobo6 fluorescence signal upon incubation with collagen I gels, at varying concentrations. Binding curves were used to extract a value for the observed equilibrium constant (k_obs) at each concentration (cf. Methods). f, Linear fit between k_obs and Rhobo6 concentrations from (e), allowing extrapolation of binding constants k_on and k_off. An apparent dissociation constant K_D of 53 μM was determined by the ratio of the two. Error bars represent 95% confidence interval for fitted k_obs values. g, Confocal microscopy of MCF10A cells labeled with Rhobo6. Expression of GFP-MUC1ΔCT was induced via addition of doxycycline. Mucin domains, which are N-terminal to GFP, were degraded enzymatically via live cell treatment with the mucinase StcE. Note that mucin overexpression in these cells induces them to lift from their growth substrate, causing a spherical appearance with no loss in viability. Contrast is normalized for each channel across experimental conditions. h, FLIM microscopy of MCF10A+GFP-MUC1ΔCT cells labeled with Rhobo6. Phasor plot (top) of lifetime distribution, with ROIs marking unbound and bound Rhobo6 population. Bottom, Intensity contrast image compared to lifetime bandpass images for each population (cf. Supplementary Table 1).

Figure 3.. Labeling of excised tissues by bathing in Rhobo6-containing media.

a, Cartoon illustrating the labeling approach. Freshly dissected or cultured tissues were labeled with 5 μM Rhobo6 for 1 h, with sample-specific media (cf. Methods). b, Viability and morphogenesis of mouse embryonic salivary glands upon incubation with Rhobo6. N = 14 glands were split into two paired groups, with each pair corresponding to glands from a single embryo. The first group was incubated with 5 μM Rhobo6 in media containing 0.5% DMSO, and the second group was incubated in media containing 0.5% DMSO, as a vehicle control. Viability and morphogenesis were assessed by counting epithelial buds every 24 h for 2 days. Paired groups were compared by paired t-tests; NS = not significant. c, Mouse embryonic submandibular salivary gland (E14) cultured ex vivo for 5 days, then labeled by bathing concurrently with Rhobo6 and 6-carboxyrhodamine 110. The latter dye differs from Rhobo6 only in that it does not contain the two ortho-aminomethylphenyl boronic acid groups, which are necessary for binding to extracellular glycans. Images were denoised (see Extended Data Fig. 4b–c for comparison of raw and denoised salivary gland images; Supplementary Table 1 reports image processing workflow for all datasets). d, Comparison of live Rhobo6 labeling to live labeling with protein-based affinity reagents against common ECM components, including fibrous collagen (CNA35), network-forming collagen (anti-collagen IV) and laminins. Glands were incubated with purified CNA35-GFP or Atto647N conjugated antibodies in solution along with Rhobo6, and imaged with a confocal microscope. Contrast not normalized. Images were denoised (cf. Supplementary Table 1). e, Freshly dissected and exsanguinated mouse pancreatic tissue, labeled by bathing with Rhobo6 (red), to highlight ECM, and Hoechst (cyan), to localize nuclei. Image shows a maximum intensity projection over a depth spanning 23 μm. Images were denoised (cf. Supplementary Table 1). f, Two-color labeling of exsanguinated adult mouse pancreatic tissue labeled by both Rhobo6 (red) and Anti-collagen-I-ATTO647N (cyan) antibody. Tissue was labeled by bathing for 1 h with both probes, and imaged with a confocal microscope. Image shows three-dimensional reconstruction of the (100 μm)³ volume.

Figure 4.. Rhobo6 distributes across mouse organs and labels the ECM upon retroorbital injection.

a, Cartoon illustrating labeling approach. Anesthetized mice were injected retroorbitally with 100 μl of a 1 mM Rhobo6 solution in PBS containing 10% DMSO. Mice were allowed to recover for 30 min on a warming pad, then euthanized by cervical dislocation. Live tissues were harvested, placed on a glass bottom dish and imaged within 2 h of dissection. b, Two photon image of a 2 mm by 2 mm area of muscle tissue (masseter). Insets show sequential crops of the original image, highlighting both macroscopic and microscopic ECM features made visible by Rhobo6 labeling. For annotations of numbered landmarks see (c). c, Individual fields of view cropped from 2 mm by 2 mm two-photon images of the indicated tissues. Numbers in yellow correspond to features consistent with histological annotations^,. Muscle (cf. (b)): (1) skeletal muscle fibers, (2) collagen-rich fascia, and (3) basal lamina surrounding myofibrils. Trachea: (4) tracheal cartilage ring, (5) submucosal layer with basement membrane, and (6) a tracheal gland encased in ECM. Pancreas: (7) longitudinal section of an interlobular duct, (8) the cross section of an intercalated duct, and (9) acinar tissue. Kidney: (10) a collecting tubule with a branching point, and (11) proximal and distal convoluted tubules. Jejunum: (12) mucus layer, (13) stratified squamous epithelial layer, and (14) villi. Tendon: (15) fascia of tertiary fiber bundle, and (16) fibroblasts. Liver: (17) entire field of view shows the fascia layer superficial to hepatocyte layer. Gallbladder: (18) longitudinal section of a capillary. Lung: (19) entire field of view shows alveolar tissue encased in ECM. All tissues, including images in (b) and (d), were acquired on the same day from 4 different mice of same strain and age (cf. Methods). Contrast not normalized across samples. d, Three-dimensional reconstructions of three tissues, from two-photon microscopy volumes. Depth color coding applied. Histological annotations are numbered in yellow. Salivary gland: (20) epithelial buds. Brain: (21) a blood vessel on the brain surface, (22) red blood cells excluded from Rhobo6 labeling within the vessel, and (23) brain tissue which is not labeled by Rhobo6, therefore appearing dark. Skin (24) collagen fibers and (25) elastin fibers. Contrast and depth-coded lookup table not normalized across samples. Images in (d) were denoised (cf. Supplementary Table 1).

Figure 5.. Intravital 2P imaging of ECM in a mouse model of mammary carcinoma.

a, Cartoon representing experimental timeline, along with intravital imaging strategy for wild-type and mammary tumor bearing MMTV-PyMT mice. b, Rhobo6 imaging with three fields of view from the same mammary gland marking the extracellular matrix surrounding normal ductal architecture. Left, Volume rendering (depth-color coding applied). Arrows indicate adipocytes and epithelial ducts. Right, A single confocal slice from the adjacent volume (red dashed plane, with Z-height indicated) illustrating Rhobo6 labeling. Contrast is not normalized. Images were denoised (cf. Supplementary Table 1). c, The same as in (b) for two individual MMTV-PyMT mice. Two FOVs are presented for mouse 1 and one FOV for mouse 2. Arrows indicate early stage and late stage carcinomas. Contrast is not normalized. Images were denoised (cf. Supplementary Table 1).