C-Laurdan imaging was performed as previously described 98 (link)–102 (link). Briefly, cells were washed with PBS and stained with 10 µg/mL C-Laurdan for 10 min on ice, then imaged using confocal microscopy on a Leica SP8 with spectral imaging at 60× (water immersion, NA= X) and excitation at 405 nm. The emission was collected as two images: 420–460 nm and 470–510 nm. MATLAB (MathWorks, Natick, MA) was used to calculate the two-dimensional (2D) GP map, where GP for each pixel was calculated as previously described 102 (link). Briefly, each image was background subtracted and thresholded to keep only pixels with intensities greater than 3 standard deviations of the background value in both channels. The GP image was calculated for each pixel using Eq.1. GP maps (pixels represented by GP value rather than intensity) were imported into ImageJ. To calculate the average PM GP, line scans drawn across individual cells. PM GP values were taken as peak GP values from the periphery of the cell, whereas internal membranes were calculated as the average of all values outside the PM peak. The average GP of the internal membranes was calculated by determining the average GP of all pixels in a mask drawn on each cell just inside of the PM.