Dg4 light source

Cells were imaged 3 days after transfection, in saline consisting of 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5 mM glucose, 20 mM HEPES buffered to pH 7.3 using NaOH. Stimulation was achieved through depolarization elicited by substitution of an additional 20mM KCl for NaCl in the bathing solution. Cover slips with cells were mounted in an imaging chamber (Warner Instruments) and viewed from beneath with conventional epifluorescence optics and an APO N 65x 1.49 NA objective optimized for fluorescence (Olympus). Image acquisition was controlled by a computer running Slidebook 4.2 (Intelligent Imaging Innovations). Imaging was carried out on an Olympus ix80 inverted microscope with integrated high precision focus drive. Fluorescence excitation was provided by a rapid switching DG4 light source (Sutter Instruments) attached by liquid light guide, and overall image acquisition rates was 4 Hz. The cooled CCD camera was a Hammamatsu ORCA R2, communicating to the host computer via firewire interface. TIRF illumination was achieved through the objective, using 488 and 536 nm lasers (Melles Griot), with fine adjustment by micrometers to optimize TIRF illumination. The depth of evanescent field was assessed using apparent size of ring-stained 1 micron beads, and geometric calculations suggest an estimate of 80–100nm.