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. 2020 Mar 19;31(7):709–723. doi: 10.1091/mbc.E19-10-0559

FIGURE 2:

FIGURE 2:

Very large data sets from ImFCS measurements are analyzed to examine spatially heterogeneous diffusion properties of plasma membrane probes, as exemplified by EGFP-GG. (A) A typical ImFCS measurement records image stack of 80,000 frames (3.5 ms/frame) from a region of interest (ROI) on the ventral plasma membrane. Representative first few images of 2 × 2 binned pixels (Px units) are shown. Representative Px unit (red box: 320 × 320 nm2) and Sv unit (green box: 1.28 × 1.28 μm2) in the image stack are shown. (B) Raw autocorrelation functions (ACFs; black) and respective fits (gray) for each Px unit using a single-component Brownian diffusion model (Eq. 1). Inset: spatial map of extracted diffusion coefficient (D) values at each Px unit of the image stack represented in A. (C) D values averaged over ROIs in 18 individual RBL cells expressing EGFP-GG; error bars are standard deviations about an average D for each cell. ROIs generally contain 400–625 Px units covering about 41–64 μm2 membrane area for each cell, yielding 10,527 total D values for 18 cells in this example. (D) Top: 10,527 D values obtained from ROIs in all cells are pooled and plotted as a normalized cumulative distribution function (CDF), which is fitted with one (Eq. 2) or two (Eq. 3) components, as indicated. The inset shows the same data for D plotted as a probability distribution function (PDF) with arbitrary binning of parameter values. Bottom: residual plots for one-component and two-component fits to CDF. (E) svFCS analysis is carried out on each Sv unit of the same raw data as depicted in A. A representative Sv unit is outlined in green, and four different sizes of observation area (Aeff) are created by pixel binning within the Sv unit (pink, red, orange, and blue boxes). (F) Linear svFCS plots (diffusion time [τD] vs. observation area [Aeff]; Eq. 5) are generated from all possible nonoverlapping Sv units (represented in A), yielding values for y-intercept (τ0) and Slope (1/Deff). Inset: spatial map of τ0 values determined from each Sv unit of an ROI represented in A.