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

摘要

a A schematic description of imaging T/APC conjugates on opposing coverslips. b Large-scale microscopy images consisting of 100 fields of view were taken of CD8+ T cells in contact with APC (T2 hybridoma) cells loaded with the activating peptide NY-ESO-1. A montage of large-scale bright-filed images and a single field is shown (top row). Image contrast of these images was adapted here for improved visibility of single cells. The PM of the CD8+ T cells was stained for αCD45 and Alexa647 (red) and the PM the T2 cells was stained using DPEE-Atto565 (green). Fluorescence images of the zoom field are shown (bottom row). Large-scale images in bright field, and in each of the fluorescence channels were routinely collected at different z-sections and are shown in Fig. S1. c Zoomed bright field and fluorescence images from the single field in panel b are shown at different heights (focal planes) relative to the cell interface. d Calcium imaging of Fluo-4 stained live CD8+ T cells (yellow arrowhead), upon encounter with the T2 cells (magenta arrowhead) loaded with the activating peptide NY-ESO-1. A representative field is shown. Yellow arrowheads show an activated cell of interest. e The intensity time-trajectory of Fluo-4 in the pointed CD8+ T cell in panel d. Magenta arrowhead indicates the time of the cell engagement with the T2 APC. f The time-dependent killing efficiency by CD8+ T cells of the T2 cells, with (blue) or without (magenta) loading with NY-ESO-1 peptide.