Xdi revolution

HEK293 cells expressing various Orai1-YFP mutants were imaged on an Andor XDI Revolution spinning-disk confocal microscope equipped with a 100× oil immersion objective. Fluorophores were excited with 515 nm (YFP) laser diodes with the intensity of laser light attenuated to 20–40%. Images were obtained at 512 × 512 pixels at an exposure of 200–500 ms per frame and a slice thickness of 0.8 µm. Images analysis was performed using ImageJ software (National Institutes of Health).

FRAP was performed on live cells housed within a 37°C chamber using a spinning disk confocal microscope (XDI Revolution equipped with a Yokogawa CSU-X1; Andor Technology). Images were acquired with a Nikon Plan Apo 100×1.49NA TIRF objective and an Andor iXon3 EMCCD camera using MetaMorph 7.7 (Molecular Devices). Three prebleach images were acquired. Photobleaching of 75% was performed for three iterations at the appropriate wavelength. Image acquisitions of fluorescence recovery were performed 5 s after initial FRAP for 100 s. Imaging intervals were then increased to every 60 s to assure complete recovery after photobleaching. The intensity within the bleached region and a nonbleached region (as a control) were first measured using Fiji/ImageJ and then plotted to obtain the t_1/2 values.