Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes Articles uri icon

publication date

  • April 2016

start page

  • 1

end page

  • 10

volume

  • 431

international standard serial number (ISSN)

  • 0022-1759

electronic international standard serial number (EISSN)

  • 1872-7905

abstract

  • Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10 mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues. (C) 2016 Elsevier B.V. All rights reserved.

keywords

  • light sheet fluorescence microscopy; tissue clearing; lymph nodes; t cell-dc interactions; optical projection tomography; cd8(+) t-cells; dendritic cell; viral-infection; cd4(+); activation; help; reconstruction; trafficking; expression