Fig. 4.

Correlative 3D light and FIB-SEM of activated T cells. Jurkat cells transfected with emerald-VAMP7 (green) were dropped onto stimulatory coverslips and fixed after 2 min a–h or 5 min i–n of activation and immunostained for pLAT (red), nucleus (blue), and plasma membrane (purple). a Images were collected of the whole cell activated for 2 min using confocal microscopy. Box 1 indicates a region with a pLAT microcluster and Box 2 indicates a region with VAMP7 vesicles. b Focused ion beam scanning electron microscopy (FIB-SEM) images were subsequently collected from the same samples. Scale bars for a and b equal 2 μm. c, f Zoomed-in FIB-SEM image of ROI 1 and ROI 2, respectively, showing a single slice. Scale bars for c and f equal 0.5 μm. d, g Zoomed-in FIB-SEM stacks corresponding to ROI 1 and 2, respectively. Corresponding FIB-SEM stacks are in Supplementary Movies 8 and 10, respectively. e, h Segmented FIB-SEM volumes corresponding to ROI 1 and 2, respectively; correspond to Supplementary Movies 9 and 11, respectively. i Images were collected through the whole cell activated for 5 min using confocal microscopy. Box 1 indicates a region with a pLAT microcluster in proximity of VAMP7 signal and Box 2 indicates a pLAT microcluster devoid of VAMP7 signal. j Focused ion beam scanning electron microscopy (FIB-SEM) images were subsequently collected from the same samples. Scale bars for i and j equal 2 μm. k, m Zoomed-in FIB-SEM image of ROI 1 and 2, respectively, showing a single slice. The arrowheads indicate subsynaptic vesicles. Scale bars for k and m equal 0.5 μm. Corresponding FIB-SEM stacks are in Supplementary Movies 12 and 13, respectively. l, n Segmented FIB-SEM volumes corresponding to ROI 1 and 2, respectively; see Supplementary Movies 14 and 15 (n = 3 cells for 2 min; 3 cells for 5 min, two independent experiments). See also Supplementary Figure 3