Widefield Microscopy of Membrane Fusion

For fusion measurement, samples were prepared in Grace BioLabs CultureWells with coverslip bottom treated with 1% Pluronics F-127 (1 h). Timeseries of fusion events were collected at 49-ms time intervals on a widefield Axio Observer 7 Inverted Microscope (Zeiss) with a ×63/1.4 NA Plan-Apochromat (oil immersion) objective. FITC was excited with a 120-W metal halide lamp (X-cite 120) with 470/40 nm excitation and 525/50 nm emission filters. Images were acquired with a Axiocam 506 mono camera (Zeiss) controlled by the Zen software (Zeiss). ImageJ was used to further format and process images, and MATLAB was used to analyze fusion events as described previously^{69 (link)}.