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Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Published on: August 23, 2007
Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes
Jun Abe1, Aleksandra J Ozga1, Jim Swoger2
1Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland.
Insights
Light sheet fluorescence microscopy enables visualization of T cell and dendritic cell interactions within lymph nodes. This technique allows for detailed spatial analysis of immune cell behavior crucial for understanding immune responses.
Area of Science:
- Immunology
- Microscopy
- Cell Biology
Background:
- Reactive lymph nodes (LNs) are critical sites for T cell activation by dendritic cells (DCs).
- The spatial organization of T cell-DC interactions at low precursor frequencies remains poorly understood.
- Light sheet fluorescence microscopy (LSFM), including selective plane illumination microscopy (SPIM), offers potential for high-resolution imaging of tissues like LNs.
Purpose of the Study:
- To establish and validate protocols for applying LSFM/SPIM to immunologically relevant tissues, specifically murine LNs.
- To assess the feasibility of visualizing and quantifying single-cell interactions between T cells and DCs within cleared LNs.
- To define parameters for analyzing T cell-DC interactions in the context of adaptive immunity.
Main Methods:
- Development of tissue clearing protocols to preserve fluorescent protein integrity for SPIM.
- Application of SPIM to optically cleared murine LNs containing adoptively transferred T cells and DCs.
- 3D reconstruction and single-cell analysis of SPIM data to quantify cell distribution and interactions.
Main Results:
- Protocols were successfully established to maintain fluorescent protein function in lymphoid tissues after clearing.
- SPIM enabled the detection and 3D spatial mapping of individual T cells and DCs within cleared murine LNs.
- Quantitative parameters were defined to characterize specific interactions between antigen-specific T cells and pMHC-bearing DCs.
Conclusions:
- LSFM/SPIM is a powerful and applicable technique for high-resolution imaging of immune cell interactions in intact lymphoid organs.
- This methodology provides new insights into the spatial dynamics of T cell priming within lymph nodes.
- The study successfully demonstrates the utility of LSFM for advancing immunological research.
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.
