Live-cell Imaging of Endothelial Cytoskeletal Dynamics

The lattice light sheet microscope located at the Advanced Imaging Center (AIC) at the Janelia Research Campus of the Howard Hughes Medical Institute (HHMI) (Chen et al., 2014 (link)) was used. HUVECs stably expressing dTomato-2xrGBD and mTurquoise2-CaaX were cultured on fibronectin-coated 5 mm round glass coverslips (Warner Instruments, Catalog # CS-5R) for 2 days. Cells were imaged at 37°C in the presence of 5% CO₂ in HEPES buffer (132 mM NaCl₂, 20 mM HEPES, 6 mM KCl₂, 1 mM MgSO₄•7H₂O and 1.2 mM K₂HPO₄•3H₂O at pH 7.4), supplemented with 1 mM CaCl₂, 0.5% Albuman (Sanquin Reagents, The Netherlands) and 1 g/l D-glucose. Illumination was undertaken using 445 nm and 560 nm diode lasers (MPB Communications), acousto-optic tunable filter (AOTF) transmittance and 100 mW initial box power and an excitation objective (Special Optics, 0.65 NA, 3.74-mm WD). Fluorescence detection was done via a detection objective (Nikon, CFI Apo LWD 25XW, 1.1 NA) and a sCMOS camera (Hamamatsu Orca Flash 4.0 v2). Point-spread functions were measured using 200 nm tetraspeck beads (Invitrogen cat# T7280) for each wavelength. Data was deskewed and deconvolved as described previously (Chen et al., 2014 (link)).