High-Speed Single-Molecule Tracking of CXCL13 in the B-Follicle

Abstract

Soluble factors are an essential means of communication between cells and their environment. However, many molecules readily interact with extracellular matrix components, giving rise to multiple modes of diffusion. The molecular quantification of diffusion in situ is thus a challenging imaging frontier, requiring very high spatial and temporal resolution. Overcoming this methodological barrier is key to understanding the precise spatial patterning of the extracellular factors that regulate immune function. To address this, we have developed a high-speed light microscopy system capable of millisecond sampling in ex vivo tissue samples and submillisecond sampling in controlled in vitro samples to characterize molecular diffusion in a range of complex microenvironments. We demonstrate that this method outperforms competing tools for determining molecular mobility of fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) for evaluation of diffusion. We then apply this approach to study the chemokine CXCL13, a key determinant of lymphoid tissue architecture, and B-cell-mediated immunity. Super-resolution single-molecule tracking of fluorescently labeled CCL19 and CXCL13 in collagen matrix was used to assess the heterogeneity of chemokine mobility behaviors, with results indicating an immobile fraction and a mobile fraction for both molecules, with distinct diffusion rates of 8.4 ± 0.2 and 6.2 ± 0.3 µm²s^-1, respectively. To better understand mobility behaviors in situ, we analyzed CXCL13-AF647 diffusion in murine lymph node tissue sections and observed both an immobile fraction and a mobile fraction with an example diffusion coefficient of 6.6 ± 0.4 µm²s^-1, suggesting that mobility within the follicle is also multimodal. In quantitatively studying mobility behaviors at the molecular level, we have obtained an increased understanding of CXCL13 bioavailability within the follicle. Our high-speed single-molecule tracking approach affords a novel perspective from which to understand the mobility of soluble factors relevant to the immune system.

Keywords: biophysics; chemokines; lymphoid tissues; single-molecule imaging; single-molecule tracking.

Figure 1

Schematic diagrams of high-speed narrowfield microscopy and the experimental system. (A) The imaging framework showing the bespoke fluorescence microscope and diagrams of image acquisition. (B) The structure of Alexa Fluor 647 labeled CCL19 and CXCL13.

Figure 2

Single-molecule stoichiometry of CCL19-AF647. (A) Tracking of photoblinking Alexa Fluor 647 (AF647): localizations and intensity over time with sample images from the acquisition. (B) Distribution of apparent CCL19 foci stoichiometry (gray) overlaid with the predicted distribution based on randomly overlapping point spread functions (blue). (C) Kernel density estimates of intensity of AF647 labeled CCL19 in collagen (solid blue line), and under heparan sulfate immobilization (solid black line); CXCL13 in collagen (dotted blue line), and under heparan sulfate immobilization (dotted black line); and bovine serum albumin in 10% Ficoll (solid red line). All traces are normalized to the primary peak for clarity (see Supplementary Material).

Figure 3

Simulations of chemokine data. (A) Sample simulation images, shown with and without Gaussian white noise added. Scale bar 1 µm. (B) Two-gamma distribution fit (red) to diffusion coefficients found from a simulation of 1.6 and 10 µm²s⁻¹ data with Gaussian white noise. (C) Histograms showing the distribution of simulated 0 µm²s⁻¹ data with (red, overlaid) and without (blue) Gaussian white noise. (D) Diffusion coefficient distribution from a simulation of 0 and 9 µm²s⁻¹ data with Gaussian white noise. Fitted populations are shown in black for the immobile, red for the mobile, and blue for the combined fit. Shaded areas indicate one SD.

Figure 4

Comparing fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single-molecule tracking on BSA-AF647 in 10% Ficoll 400. (A) Single-molecule tracking: simplified schematic of the stages in tracking and the resulting fit with shaded regions indicating error bounds of one SD. (B) FRAP: schematic of technique, profile of bleached region in an immobilized sample, and example fluorescence intensity recovery trace. (C) FCS: schematic of the confocal volume, example section of intensity fluctuation trace, and correlation curve.

Figure 5

Single-particle tracking of chemokines in collagen. Second-harmonic imaging microscopy (SHIM) of collagen network in (A) 2D and (B) 3D. (C) Representative consecutive submillisecond images of chemokines in collagen. (D,E) Fitted diffusion coefficient distribution of CXCL13-AF647 and CCL19-AF647 showing mobile and immobile components in a collagen matrix with (F) just the fitted high-mobility diffusion coefficient distributions of CXCL13-AF647 (cyan) and CCL19-AF647 (magenta) (shaded areas indicate one SD).

Figure 6

Confocal microscopy quantification of CXCL13-AF647 binding to lymph node follicles. (A) Schematic diagram of approximate locations of B-cell follicles in a wild-type murine lymph node. (B) Exemplar confocal microscopy images of CXCL13-AF647 and BSA-AF647 binding to lymph node tissue follicles (B220 + regions of lymph node tissue sections), and control with only B220 staining. (C) Quantification of the total fluorescent intensity for a fixed size imaging plane within a lymph node follicle. Each data point represents a distinct follicle.

Figure 7

Single-molecule analysis of CXCL13-AF647 in tissue. (A) Intensity average image of image acquisition to show autofluorescent extracellular matrix (ECM) in B220-stained B-cell follicle with no added chemokine. (B) Areas of (A) identified as ECM by segmentation with overlaid track localizations colored orange. (C) Intensity average image of image acquisition to show autofluorescent ECM in B220-stained B-cell follicle with added chemokine. (D) Areas of (C) identified as ECM by segmentation with overlaid track localizations colored by location on ECM (blue) or in the interstitial spaces between cells (cyan). (E) Comparison of diffusion coefficients of localizations in ECM locations in the presence (blue) and absence (orange) of CXCL13-AF647 (F) Comparison of diffusion coefficients for the ECM (blue) and chemokine (cyan) populations when tracking CXCL13-AF647 in lymph node tissue shown. (G) Distribution and fit of chemokine diffusion coefficients of CXC13-AF647 in tissue sections, shaded area indicates one SD.