Fig. 4: ROCS microscopy of mast cell degranulation.

a Mouse peritoneal mast cells coated with IgE are triggered by DNP-HSA. 10 s after stimulation the degranulating mast cells are recorded in parallel with TIR-ROCS at 100 Hz and with TIR-F at 5 Hz over 60 s (12 s shown). b Difference images reveal significant changes within only 10 ms with ROCS (left), but hardly over 200 ms with Avidin-FITC fluorescence (right). A strong drop in ROCS-intensity (black area in the region of interest ROI 2) suggests a degranulation channel (cavity), which opens within < 10 ms. c The magnified ROI 1 shows cell surface areas imaged with TIR-ROCS, which turn into degranulation pores visible as black patches. Further cavities are visible. d Sketched cross-section of a mast cell on coverslip within the TIR illumination area. e A minimum projection (selection) from 150 ROCS difference images (4.0 s–5.5 s) reveals differently shaped degranulation pores. f Kymograph of 1000 ROCS difference images along the line in figure b reveal the opening of two adjacent pores within < 10 ms shown by the black lines (blue arrows). g Kymographs along the line scan in figure a: Top: the TIR-F channel shows continuous Avidin-FITC uptake of the vesicles in the pore, the TIR-ROCS reveals a stepwise reduction of intensity from disappearing light-scattering material. h cartoon pointing out the possible scenario in the mast cell periphery after stimulus: changes in fluorescence become apparent after decrease of ROCS signals. i Degranulation and release of one granule vesicle in lateral direction within TIR-region. Vesicle dynamics is visible both with 5 Hz fluorescence and 100 Hz ROCS imaging. Inset: ROCS image projection over 2 s (200 frames) indicating the subsequent steps 1…4 of granule release. j TIR-F signals of degranulation are noisy and become invisible after > 1 s, while TIR-ROCS signals enable detailed kymographs both in intensity and difference images (bottom). Source data are provided as a Source Data file.