Fig. 4: High labeling density enables the visualization of membrane protein topology of Jurkat T cells on activating glass surfaces and material transfer across the membrane through microvesicles and phagocytosis.

a Representative DeepSIM volumetric superresolution image of a pan-membrane-protein labeled Jurkat T cell on a non-stimulating surface coated with poly-L-lysine (PLL). b Zoomed-in views of the regions indicated in (a) showing T cell microvilli (i) and membrane protein assemblies (ii). c Representative volumetric superresolution image of a pan-membrane-protein labeled Jurkat T cell on a stimulating surface coated with anti-CD3 (OKT-3) antibody. d Zoomed-in views of the regions indicated in (c) showing membrane ruffles (i) and membrane protein assemblies (ii). e A zoomed-in volumetric superresolution image of a pan-membrane-protein labeled Jurkat T cell incubated for 1 h on a stimulating surface. Bottom-up view. f Orthographic views of the indicated EV in (e) demonstrating volumetric membrane protein distribution. g The cross-sectional view of the indicated EVs in (e). The diameter at the widest z-position is shown on each vesicle. i: z = 2 μm, ii: z = 4 μm, and iii: z = 6 μm. h Distribution of the extracellular vesicle (EV) diameters. n = 19 EVs. i Zoomed-in volumetric fluorescent view of the Dynabead internalization across the membrane indicated in Supplementary Fig. 9c. White arrows indicate the internalized Dynabead with co-internalized membrane proteins. Yellow arrows point to a Dynabead that is not internalized. The complete image sequence is shown in Supplementary Movie 15. Scale bars: 10 μm (a, c), 5 μm (b, d and i), 2 μm (e), and 1 μm (f, g).