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Lookup NU author(s): Dr Adam WollmanORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
© 2018 Miller, Cosgrove, Wollman, Taylor, Zhou, O'Toole, Coles and Leake.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.
Author(s): Miller H, Cosgrove J, Wollman AJM, Taylor E, Zhou Z, O'Toole PJ, Coles MC, Leake MC
Publication type: Article
Publication status: Published
Journal: Frontiers in Immunology
Year: 2018
Volume: 9
Online publication date: 22/05/2018
Acceptance date: 30/04/2018
Date deposited: 10/02/2020
ISSN (electronic): 1664-3224
Publisher: Frontiers Research Foundation
URL: https://doi.org/10.3389/fimmu.2018.01073
DOI: 10.3389/fimmu.2018.01073
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