Fig. 2

Emission anisotropy of GFP-LFA-1 fusions. a Schematic of emission anisotropy TIRF microscopy²⁷. The transition dipole of GFP has an excitation dipole (green) that is very close in orientation to its emission dipole (red)^28,29. I _II and I _⊥ are emission intensities parallel and perpendicular to the direction of polarized excitation. b–d Schematics showing a cell and integrins in same microscope xy plane as in a and different outcomes in emission anisotropy. The excitation (electric) field of polarized light is shown as a blue wave. b. Outcomes using a constrained integrin-GFP fusion. Depending on the orientation of integrins within pixels (1–3) or regions of interest (ROI), the emission anisotropy will change as indicated. c, d Outcomes with aligned integrins with constrained c or unconstrained GFP d. e Representative images from movies of Jurkat T cells stably expressing GFP-LFA-1 fusions migrating on ICAM-1. Each pair of panels (scale bars are 5 µm) shows total GFP fluorescence intensity (upper) and anisotropy (lower, color scale to right). f Emission anisotropy of GFP-LFA-1 fusions, averaged over Jurkat T cells migrating in random directions, in at least five independent experiments. Box plots show the full range (whiskers) of observations with median as line and 25–75 percentile range boxed. Kruskal–Wallis test with multiple comparison correction gave the indicated P values. N (number of cells) from left was 18, 22, 17, 37. *p < 0.05; **p < 0.01; ****p < 0.0001