Super-resolution volumetric imaging of the immune synapse using dSTORM and lattice light-sheet microcopy

Abstract

T cells, integral components of the adaptive immune system, are crucial for antigen recognition within the body. Activation of T cells is initiated when the T cell receptor (TCR) binds to a major histocompatibility complex (MHC) loaded with an antigenic peptide (pMHC) on the surface of antigen-presenting cells (APCs). The topography of the T cell-APC synapse and spatial distribution of proteins within it are vital for effective antigen recognition. However, achieving high-resolution imaging of the immune synapse has been challenging due to its complex geometry and orientation relative to the optical axis. Commonly, total internal reflection fluorescence (TIRF) microscopy has been used in conjunction with functionalized supported lipid bilayers (SLBs) to study the T cell activation. However, this method may introduce artifacts due to the simplified nature of the system and the rigidity of SLBs. In this study, we present an innovative approach that combines three-dimensional single-molecule localization microscopy (dSTORM) with lattice light-sheet microscopy. This technique allows us to achieve high spatial resolution and volumetric imaging of entire cells. As a proof of concept, we utilized the nuclear pore complex (NPC) to benchmark our super-resolution imaging method and successfully resolved its structure. Applying this method to T cells promises to provide deeper insights into the mechanisms of T cell activation, potentially advancing our understanding of immune responses.