3D-SIM microscopy

Imaging was performed using a Zeiss Elyra PS1 system. 3D-SIM data was acquired using a 63 × 1.4NA oil objective. 488 nm, 561 nm, 642 nm, 100 mW diode lasers were used to excite the fluorophores together with respectively a BP 495–575 + LP 750, BP 570–650 + LP 750 or LP 655 emission filter. For 3D-SIM imaging a grating was present in the light path. The grating was modulated in 5 phases and 5 rotations, and multiple z-slices were recorded with an interval of 110 nm on an Andor iXon DU 885, 1002 × 1004 EMCCD camera. Raw images were reconstructed using the Zeiss Zen software.

Reconstructed 3D-SIM images were analyzed with imageJ⁴⁷ extended in the FIJI framework⁴⁸. Particles larger than 10 pixels were detected and marked as region of interest (ROI) and mean ADGRA1 or PLEKHA5 signals inside the ROIs were measured. A HOMER1 particle was counted as positive for ADGRA1 or PLEKHA5 if their mean intensity was more than three times above their respective local backgrounds.