K-Space Image Correlation Spectroscopy of Quantum dot Labeled T Cell Receptors Characterizes their Nanoscale Clustering in Living Cells

Changes in distribution of membrane receptor organization are used by cells to modulate the dynamic range of their responses to environmental cues. Consequently, it is important to develop experimental methods that can accurately measure receptor transport and aggregation. We used quantum dot (QD) labeling of T cell receptors (TCR) and a recently developed technique, k-space image correlation spectroscopy (kICS) to characterize TCR state as a function of cell differentiation. We developed kICS to measure transport coefficients of fluorescently labeled membrane proteins while taking into account nanoparticle emission blinking. We use kICS to measure T cell receptor (TCR) aggregation in live cells by characterizing quantum dot (QD) blinking and distribution on the cell surface. 2C TCR transgenic cells in culture were observed from the naive state to 12 days after activation by antigen. Cells were labeled with biotin-MHC/peptide to bind the TCR with relatively high avidity, followed by streptavidin-quantum dots. They were then imaged on an emCCD-equipped microscope and kICS analysis was applied. Spatial intensity fluctuations in an image measured the clustering of receptors on the 100s of nm length scale. Changes in the intensity correlation function of the blinking QDs characterized clustering on the 10s of nm length scale. We also used kICS to measure changes in TCR diffusive transport. When T cells exhibited maximum activity 3-4 days after exposure to antigen, the degree of their TCR aggregation on both length scales was significantly higher than that of naive cells, while TCR diffusion was a minimum. This new technology has powerful applications as it can be applied to just a few cells and we will show that it is able to detect changes in receptor organization of cells in vivo.