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Next using Labview, the number of Cav1 structures having proximal Cavin1 structures was calculated (distance between CMCav1 and CMCavin1?[http://www.selleckchem.com/MEK.html http://www.selleckchem.com/MEK.html] were performed with a time interval of 1 or 0.5 s. During analysis, for any time point, first the background fluorescence was subtracted, next a normalization [http://en.wikipedia.org/wiki/Autophagy Autophagy] was done to compensate for photo-bleaching (with intensity of a region-of-interest (ROI) covering a large part of the cell), and then a normalization with the pre-bleach intensity was done. Mobile fractions (A) were estimated by fitting the averaged FRAP curves to a standard recovery equation (I(t)?= A(1-exp(-t/��)) where I(t) is the recovered intensity at time t and �� is the half-life for recovery. Lines show fit for the curves for iso- and hypo-osmotic conditions. For checking the statistical significance, A for each cell was derived from individual fits and the mobile fractions (A) for iso-osmotic conditions compared with that of hypo-osmotic conditions. Cells were always [http://www.selleckchem.com/products/Etopophos.html learn more] maintained at 37��C during imaging. Image analysis for all experiments was done using Labview 8.5 and Vision 8.5 (National Instruments, Austin, TX). FLIM microscopy experiments were performed as described previously ( Hill et?al., 2008). RICM was performed with an inverted microscope (Axiovert 200, Carl Zeiss, Germany) equipped with an EMCCD camera (CoolSnap, Roper Scientific, NJ) using a 100x objective. A HBO mercury lamp and an interference filter (��?= 546?nm, ����?= 5?nm) was used for creating the interference image. Confocal images of Cav1-EGFP and Cavin1-mCherry in a z-section of width 0.4?��m around the equatorial plane of PMS were analyzed for colocalization by first fitting an ellipse (close to a circle) to the membrane contour and finding the fluorescence profile of both Cav1 and Cavin1 along the membrane. From these fluorescence profiles, peaks consisting of at least 5 data points and having an intensity of at least 1 standard deviation above the mean fluorescence were detected as peaks. Next, colocalization of Cav1-EGFP and Cavin1- mCherry was quantified by counting the peaks that overlap within 2 pixels (1 pixel?= 0.055?��m). Several confocal z-planes at the equatorial region of the PMS were separately analyzed to find the average number of colocalized structures.