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.
Highlights
Within the immune system, soluble factors such as chemokines, cytokines, and growth factors drive graded responses to extracellular signals, regulating processes including immune cell recruitment at sites of infection [1], lymphoid tissue formation [2, 3], and the maturation of adaptive immune responses [4]
To enable precise localization and tracking of rapidly diffusing biomolecules, we modified the optical path of a standard inverted epifluorescence microscope (Figure 1A) to implement a broadband laser whose output was selectable over wavelengths ~400–2,000 nm (Figure S1 in Supplementary Material), spanning the excitation spectra of visible-light and near-infrared fluorophores; the beam was de-expanded using a series of lenses to generate a narrow illumination field of ~12 μm full-width at half maximum (FWHM) which could be switched from epifluorescence into total internal reflection fluorescence (TIRF) by controllable translation of a lens, TIRF was not used in this work
We have demonstrated the application of a high-speed single-molecule tracking microscopy system that is compatible with traditional widefield light microscopes
Summary
Soluble factors such as chemokines, cytokines, and growth factors drive graded responses to extracellular signals, regulating processes including immune cell recruitment at sites of infection [1], lymphoid tissue formation [2, 3], and the maturation of adaptive immune responses [4]. Elastic and interferometric scattering can overcome poor fluorophore photophysics to enable rapid sampling; they either use relatively large probes that exhibit steric hindrance, or achieve poor specificity in heterogeneous sample environments unless used in conjunction with fluorescent labeling [6,7,8,9,10] Scanning fluorescence methods such as stimulated emission depletion microscopy (STED [11] are limited to ~1 Hz typical frame rates with faster imaging up to ~1,000 Hz possible by trading image quality [12]; MINFLUX imaging [13] can operate at 8,000 localizations per second in bacterial cells, but only tracks one molecule at a time, while widefield approaches such as fast variants of photoactivatable localization microscopy (PALM) [14] and stochastic optical reconstruction microscopy (STORM) [15]
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