Life 888

Live imaging was performed using a custom-built inverted spinning disk confocal microscope (3i imaging systems; model CSU-W1) attached to an Andor iXon Life 888. Image acquisition was controlled by SlideBook 6 software. Images were acquired with a Plan-Apochromat 63x/1.4 NA. Oil objective, M27 with DIC III prism, using a CSU-W1 Dichroic for 488/561 nm excitation with Quad emitter and individual emitters, at laser powers 1.1 mW (488 nm) and 0.8 mW (561 nm) for photoactivation and Vapb OE (Vapb-emerald) experiments; and laser powers 2 mW (488 nm) and 2.7 mW (561 nm) for spine structural plasticity measurements. During imaging, the temperature was maintained at 32 °C using an Okolab stage top incubator with temperature control.

Fluorescence images and movie files were obtained using microscope (Olympus IX73) equipment with total internal reflection fluorescence (TIRF) illumination and spinning‐disk confocal imaging mode (Yokogawa W1). A 60× oil‐immersed objective (1.50 N.A., Olympus) and a back‐illuminated EMCCD camera (Andor iXon Life 888) were used. The pixel size is 0.2166 µm. To maintain cell physiology, an on‐stage incubator (OKO Lab) was installed on the microscope to maintain 37 °C and 5% CO₂ for cells during imaging. To track the movements of fluorescent particles, highly inclined and laminated optical sheet (HILO) imaging was performed by properly decreasing the incident angle of the laser. A 561 nm laser was used to excite the QD, 70‐kD, and 2000‐kD dextrans, Qtracker, 100 and 500‐nm fluorescence beads. The movements of QDs and 2000‐kD dextrans in cells were acquired as movie files with a total of 1 min at 30 ms intervals, the 70‐kD dextrans were recorded with a total of 30 s at 10 ms intervals, and the endocytic fluorescence beads were recorded with a total of 3 min at 100 ms intervals. To track the movements of SGs, the spinning‐disk confocal mode was applied, with recording for 3 min at 100 ms intervals. A 488 nm laser was used to excite EGFP‐G3BP1.

Arteries were incubated in a loading solution containing a fluorescent Ca²⁺ indicator, Cal-520 acetoxymethyl ester (Cal-520/AM; 5 µM), 0.02% Pluronic F-127, and 0.35% dimethyl sulfoxide in PSS for 30 min (37 °C). All Ca²⁺ measurements were carried out in PSS. Cal-520 was excited with 488-nm wide‐field epifluorescence illumination provided by a light-emitting diode illumination system (PE-300Ultra, CoolLED) and imaged using an EMCCD camera (13-µm pixel size iXon Life 888; Andor), through a 16× (water immersion; numerical aperture of 0.8; Nikon CFI75) objective lens or 40× (water immersion; numerical aperture of 0.8; Nikon CFI Apo) objective lens. Fluorescence emission was recorded at 10 Hz. Fluorescence illumination was controlled, and images (16-bit depth) were captured by µManager.
Ca²⁺ imaging recordings were analyzed using custom FIJI macros and custom analysis software written in the Python 2.7 programming language (5 (link), 24 (link)). ROIs were automatically generated for each endothelial cell so that a direct comparison of the Ca²⁺ activity in each cell between various pharmacological applications could be made (24 (link), 47 (link)).