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

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 0.5-10mm 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.

Antigen Availability and DOCK2-Driven Motility Govern CD4+ T Cell Interactions with Dendritic Cells In Vivo

“…Samples were dehydrated in 100% MeOH and cleared in benzyl alcohol-benzyl benzoate (1:2 ratio) overnight. Imaging was performed using a customized selective plane illumination microscopy (SPIM) setup equipped with five lasers (405, 480, 561, 593.5, and 635 nm), five bandpass filters (490/40, 525/50, 593/46, 628/30 and 670/30 nm), a 2.53 light sheet objective (NA 0.07), and a 53 detection objective (NA 0.12), as described (55). Volocity or Imaris software was used for the reconstruction of volume-rendered threedimensional images, the detection of transferred cells, and distance measurements between T cells and their closest DCs.…”

Advances in Ex Situ Tissue Optical Clearing

“…Recent progress in TOC and imaging opens new avenues to study cells phenotype and distribution within transparent, entire intact lymph nodes, an approach called histocytometry. Until now FocusClear, CUBIC, SeeDB, CLARITY, BABB, and uDISCO were used for lymph nodes clearing, with one study describing a completely new method for their clearing, C e 3D . C e 3D is the first approach to utilize N ‐methylacetamide with histodenz for TOC, a solution selected from 32 tested possible OCAs.…”

“…Abe et al . tested BABB, SeeDB, Clear T2 , and CUBIC clearing compatibility with lymph nodes, concluding CUBIC to be superior over BABB (which caused a loss of congenital GFP signal) and SeeDB and Clear T2 (both of which led to insufficient transparency).…”

Toolbox for In Vivo Imaging of Host–Parasite Interactions at Multiple Scales

“…Altogether, tissue clearance methods must satisfy three basic criteria: a) all tissues must be efficiently cleared, b) cellular and sub-cellular structures must be adequately preserved, and c) tissue clearance must be compatible with fluorescence detection. Current techniques include the use of organic solvents [50][51][52][53], water [47,54,55], and electrophoresis-based protocols [56,57]. A summary of relevant methods for tissue clearance is…”

“…The first commercial devices were made available and an open source, custom built-version named OpenSPIM was produced [64,65]. OPT and/or LSFM have been used to image multicellular culture models [66][67][68][69], zebrafish [70], sparse cell populations [71], the development of plants [72], living embryos and gene mapping [58,73], as well as multiple rodent organs in health and disease conditions [50,53,74,75]. OPT and LSFM were both successfully used to obtain detailed insight of the anatomy of the flight musculature of a Drosophila fly, its nervous and digestive systems, and ß-galactoside activity at whole-body level [76,77].…”