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

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.