Application suite las af software

Mid-exponential-phase HyPer reporter cells were pelleted, washed twice with phosphate-buffered saline (PBS), resuspended in 100 μl of PBS, and exposed to air in the dark for 30 min. Forty microliters of cells was placed on a Polysine microscope slide (25 by 75 by 1 mm; Thermo Scientific, Waltham, MA), covered with a Fisher-brand microscope glass coverslip (diameter, 15 mm; thickness, 0.13 to 0.17 mm; Thermo Scientific), and then visualized under a confocal laser scanning microscope (Leica model TCS SP8; Leica Microsystems, Buffalo Grove, IL, USA). Excitation was provided at 488 nm, with emission being collected from a wavelength range of 500 to 530 nm (32 (link), 56 (link)). For each sample, at least 5 fluorescent and differential interference contrast (DIC) images were captured. The fluorescence intensities of 25 regions of interest (ROI), with each ROI containing 5 cells, from each sample were measured using Leica Application Suite (LAS) AF software. For images with fluorescence that was too weak, the ROI in the corresponding DIC images was framed, and the fluorescence was measured in the same ROI in the fluorescence image. The average fluorescence intensities of 25 ROIs were calculated and are expressed in arbitrary units (a.u.) per ROI ± standard deviation.