High-speed single-molecule tracking of CXCL13 in the B-Follicle

Research output: Contribution to journalArticlepeer-review


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
sub-millisecond 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 µm2s-1 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 a 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.
Original languageEnglish
Article number1073
Pages (from-to)1073
Number of pages15
JournalFrontiers in immunology
Early online date22 May 2018
Publication statusPublished - 22 May 2018

Bibliographical note

Funding Information:
This work was supported by the Biological Physical Sciences Institute (BPSI), MRC grants MR/K01580X/1 (to PT and ML), MC_PC_15073 (MC and ML), and BBSRC grant BB/N006453/1 (AW and ML). JC is supported by a studentship from the Wellcome Trust 4-year PhD programme (WT095024MA): Combating Infectious Disease: Computational Approaches in Translation Science. AW was supported by the Wellcome Trust [ref: 204829] through the Centre for Future Health (CFH) at the University of York, UK. The authors thank Jo Marrison and Andrew Leech (Bioscience Technology Facility, University of York) for technical assistance with FCS and FRAP microscopy, and for SEC-MALLs, respectively, Chris Power (Carl Zeiss Microscopy) for help with FCS, and Anne Theury for providing lymph node tissue sections.

Publisher Copyright:
© 2018 Miller, Cosgrove, Wollman, Taylor, Zhou, O'Toole, Coles and Leake.


  • Algorithms
  • B-Lymphocytes/immunology
  • Biomarkers
  • Cell Tracking/methods
  • Chemokine CCL19/genetics
  • Chemokine CXCL13/genetics
  • Collagen/metabolism
  • Humans
  • Image Processing, Computer-Assisted
  • Lymph Nodes/metabolism
  • Single Molecule Imaging/methods
  • Spectrometry, Fluorescence/methods

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