Airyscan 2 module

Airyscan imaging of T-cell and SLB was performed on a Zeiss Axio Observer.Z1 LSM 980 confocal laser-scanning microscope equipped with an Airyscan 2 module (Zeiss, Oberkochen, Germany) consisting of 32 concentrically arranged GaAsP PMT detectors and 2 MA-PMT channels. The acquisition was performed using the Airyscan super-resolution (SR) and best signal Smart Setup and a C Plan-Apochromat ×63/1.4 NA Oil DIC magnification objective. Illumination was provided by a Solid-State Light Source Colibri 7 LED lamp and by Diode lasers at 639 nm, 594 nm, and 488 nm with 0.4% laser power and 850 V detector gain for all channels. The imaging field was defined using a 6.0× scan zoom (crop area) and a Z-coverage spanning the totality of the synaptic cleft as defined by WGA staining. The final acquisition settings included a sequential acquisition in the order 647/594/488, a frame size of 528 × 528 px, a pixel time of 7.95 µs, a pixel size of 0.043 × 0.043 × 0.16 µm, and a doubled pixel sampling with bidirectional mean intensity averaging of acquisition lines. Time-lapse imaging was performed using ×20 magnification and the super-resolution SR4Y mode and frames were acquired every 30 s. Analyses were performed using the ZEN 3.2 system blue edition (Carl Zeiss Microscopy GmbH) and Fiji v2.1.0/1.53c (build 5f23140693)^{73 (link)}.

A Zeiss LSM 980 with Airyscan 2 module was used for detailed high-resolution confocal imaging of fixed samples, using a Plan-Apochromat 40x NA 1.3 oil DIC objective (Zeiss, 420762-9800-000) and a voxel size of 0.08 × 0.08 × 0.17 μm to capture Z-stacks. For all images, Multiplex SR-8Y settings were used and a GaAsP-PMT detector was used as a detector. Hoechst was excited using a 405 nm laser with 4% laser power. GFP was excited using a 488 laser with a laser power of 0.9%. Texas Red Phalloidin was excited with a 561 nm laser using 1.5% laser power. Alexa Fluor 647 was excited with a 639 nm laser with 1.8% laser power. To quantify filopodia density, a spot analysis on the ICAM-1 channel was performed in Imaris version 9.7.2. The estimated diameter used in this spot analysis was 0.6 μm. Spots were manually filtered based on quality, ensuring only filopodia and no background was measured. Here, we excluded junctional ICAM-1, either by negatively filtering using the VE-cadherin channel if there was one, or manually if there was not. Then, the total number of spots/filopodia was divided by the total surface imaged to create a filopodia/μm² parameter. In the overexpression experiments, we calculated this parameter for both the transduced and non-transduced ECs separately by filtering spots based on the GFP-intensity at the same spot.

MDCK cells overexpressing Syt13-Venus were cultured on μ-slide 8 well chambers (Ibidi, 80821). Cells were starved for 30 min in serum-free medium and were incubated with LysoTracker Red DND-99 (Invitrogen, L7528; 10:1000) to label lysosomes. After chasing with prewarmed growth medium for 60 min, time-lapse imaging was performed on a Zeiss LSM880 inverted confocal microscope with AiryScan2 module. Images of cells were acquired in time intervals of 350 msec using AiryScan2 Fast Mode.