Adhering interacting cells to two opposing coverslips allows super-resolution imaging of cell-cell interfaces

Abstract

Cell-cell interfaces convey mechanical and chemical information in multicellular systems. Microscopy has revealed intricate structure of such interfaces, yet typically with limited resolution due to diffraction and unfavourable orthogonal orientation of the interface to the coverslip. We present a simple and robust way to align cell-cell interfaces in parallel to the coverslip by adhering the interacting cells to two opposing coverslips. We demonstrate high-quality diffraction-limited and super-resolution imaging of interfaces (immune-synapses) between fixed and live CD8+ T-cells and either antigen presenting cells or melanoma cells. Imaging methods include bright-field, confocal, STED, dSTORM, SOFI, SRRF and large-scale tiled images. The low background, lack of aberrations and enhanced spatial stability of our method relative to existing cell-trapping techniques allow use of these methods. We expect that the simplicity and wide-compatibility of our approach will allow its wide dissemination for super-resolving the intricate structure and molecular organization in a variety of cell-cell interfaces.

Nanoscale CAR Organization at the Immune Synapse Correlates with CAR-T Effector Functions

“…We used our recently developed assay for super-resolution imaging of cell–cell interfaces as they adhere to opposing coverslips [ 14 ]. Briefly, T (or CAR-T) cells and target cells are adhered to two opposing surfaces that are brought into contact, while spherical beads serve as physical spacers between these surfaces.…”

“…Briefly, T (or CAR-T) cells and target cells are adhered to two opposing surfaces that are brought into contact, while spherical beads serve as physical spacers between these surfaces. Importantly, there was no interference with intact cell activation [ 14 ]. Here, we followed the interaction of CD138-CAR-T cells (CD138-CAR) as they engaged target cells that exclusively express CD138.…”

“…We employed single- and two-color single-molecule localization microscopy (SMLM) to resolve individual molecules on the surface of the CAR-T cells, as they engaged either on-target or off-target cancer cells. Our imaging further included a technique, recently developed in our lab, for aberration-free SMLM imaging of the interface between the cells (fixed or alive) with resolution down to ~20 nm [ 14 ]. We discovered that CARs showed significant self-clustering that was enhanced in interfaces with on-target cells (SKOV-3, CAG, FaDu) relative to negative cells (OVCAR-3).…”