Celltirf 4line excitation system

Live-cell fluorescent imaging of pHluorin-expressing neurons was carried out under the same conditions as iGluSnFR imaging. Briefly, images were acquired on an Olympus IX83 inverted microscope equipped with a cellTIRF 4Line excitation system using an Olympus 60×/1.49 Apo N objective and an Orca Flash4.0 CMOS camera (Hamamatsu Photonics). This microscope runs Metamorph software with Olympus 7.8.6.0 acquisition software from Molecular Devices. The imaging media was extracellular fluid with 2 mM CaCl₂. Single image planes were acquired with 500-ms exposure using a white organic light-emitting diode with standard green fluorescent protein filters. Images were collected once a second for 3 min. A stimulation train was started 9 s into imaging. The trains (200 stimuli in 10 s [20 Hz]) were triggered by a Grass SD9 stimulator through platinum parallel wires attached to a field stimulation chamber (Warner Instruments; RC-49MFSH). All biosensor imaging experiments were performed at 32 to 34 °C. The environment was controlled by a Tokai incubation controller and chamber.

Preparation of flow cells for single-molecule experiments was performed as described previously^{69 (link)}. Purified trans-SNARE NDs (syntaxin1a labeled with cy3, 0.5 µM) were incubated with C2AB (labeled with cy5, 1 µM) and complexin I (labeled with Alexa Fluor 488, 1 µM) at RT for 10 min, and diluted to 10 pM before injection into flow cells. Unbound protein was washed out and samples were imaged in a buffer consisting of (in mM): 1 Trolox, 0.5 CaCl₂, 100 KCl, and 25 HEPES pH 7.4, and an oxygen scavenging system (1% glucose, 1 mg/ml glucose oxidase, and 0.02 mg/ml catalase). Single-molecule imaging was performed using an Olympus IX83 inverted microscope equipped with a cellTIRF-4Line excitation system, a 60 × /1.49 Apo N objective (Olympus), and an Orca Flash4.0 CMOS camera (Hamamatsu Photonics, Skokie, IL). The following excitation filter sets: 488 nm, 590 nm, and 640 nm, were used to collect signals from Alexa Fluor 488, cy3, and cy5, respectively. Images were acquired using Metamorph and Olympus 7.8.6.0 (Molecular devices; Sunnyvale, CA), and adjusted for presentation in ImageJ.