Las af image acquisition software

To determine apical membrane eviction rates, W4 cells were transfected with Dendra-Cdc42(HVR), polarized, and imaged at 37°C in Hepes-buffered (pH = 7.4) Leibovitz’s L-15 medium (Invitrogen). Photoconversion and imaging were performed using an SP8x microscope (Leica) equipped with a temperature- and CO₂-controlled chamber using a 63× objective (HC Plan Apochromat 63×/1.40 oil) with LAS AF image acquisition software (Leica). Photoconversion of the Dendra fluorophore was performed with a pulse of 405-nm laser, and imaging was performed with a 1.5-s frame rate. To generate a fluorescent decay trace, the ratio of photoconverted (red) signal in the brush border and the total amount of red signal in the cell were determined for each time point. Moving regions of interest were drawn to correct for cell movements during imaging. Ratios were normalized and multiple traces were averaged to generate a mean fluorescence decay trace. Single exponential curve fitting was performed on the mean decay traces using MATLAB software with the general formula: f(x) = a · e^{(−b · x)} + c. A mean half-life was determined using the fitted curve. For statistical analyses, half-lives of the individual decay traces were determined and compared using an independent samples t test in SPSS with a p-value <0.05 as a cutoff for significance.