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. Author manuscript; available in PMC: 2022 Jun 30.
Published in final edited form as: Nat Immunol. 2018 Apr 16;19(5):487–496. doi: 10.1038/s41590-018-0092-4

Figure 3. PA/FCS measurements exclude the existence of short-lived higher order TCR structures.

Figure 3

(a) Scheme illustrating how T-cells quantitatively decorated with AS635P-H57-scFV and in contact with a lipid bilayer containing ICAM-1 were subjected to fluorescence correlation spectroscopy experiments combined with photon-antibunching measurements. A pulsed laser beam with a temporal width much shorter than the fluorescence lifetime was focused as a diffraction limited spot onto the T-cell plasma membrane touching the SLB as a means to probe the number of molecules moving in a correlated manner. Coincident photon pairs originating from TCR/CD3 complexes moving in an uncorrelated fashion are time lag time-independent, whereas coincident photon pairs resulting from fluorophores diffusing in a correlated manner and corresponding to higher order TCR-structures are lag-time dependent. The autocorrelation of coincident photon pairs for two different lag-times (here: from 0 ns to 2 s) hence reveals to the oligomeric state of the diffusing molecule.

(b, left panel) Shown are the measured normalized autocorrelation functions (ACFs) of diffusing, T-cell associated AS635P-H57-scFV (upper graph), AS635P-KJ25-Fab (center graph) and AS635P-KT3-scFV (bottom graph). The measured ACF (blue) was fitted with a 2D diffusion function with two components and one triplet state (red dashed line). The average diffusion yielded 0.034 ± 0.014μm2/s (AS635P-H57-scFV, n = 7 cells).

(b, right panel). Fitting respective ACFs at 0 s (blue), the 1st period (100 ns for H57 and 400 ns for KJ25 and KT3) (green) and 2 s (red) lag time yielded indicated values for the number of emitters per freely diffusing TCR/CD3 complex.

(c) Comparison of the average number of emitters for the three fluorescent probes diffusing either in solution (H57-scFV: 1.17 ± 0.01 with n = 11 measurements; KJ25-Fab: 1.26 ± 0.01 with n = 12 measurements; KT3-scFV: 1.14 ± 0.01 with n = 11 measurements) or when bound to plasma membrane-resident TCR/CD3 complexes (H57-scFV: 1.09 ± 0.08 with n = 7 cells; KJ25-Fab: 1.14 ± 0.08 with n = 7 cells; KT3-scFV: 1.86 ± 0.22 with n = 8 cells). Error bars represent the standard error of the mean.