Figure 1.
Schematic illustrating extended kICS method application. (A) An ROI and sliding time window are first chosen from an image series. Intensity fluctuations are computed locally in time, according to the chosen time-window size, to mitigate the effects of photobleaching. (B) The 2D spatial Fourier transforms of the intensity fluctuations are first computed in each frame, then this k-space ROI frame stack is autocorrelated in time. A circular averaging of the autocorrelation is also calculated when the dynamics of the system are isotropic. Nonuniformly distributed, immobile blinking fluorophore populations do not systematically affect the fluctuation-defined autocorrelation. (C) Computed ACF (points) and simultaneous fits (lines) over five time lags. (D) The process of computing the ACF and fitting is rapid (order of seconds for 64 × 64 pixel image series with 2048 frames) and outputs; the diffusion coefficient (D), the sum of the photoblinking rates (K), the fraction of time spent in the on-state (ρon), and the fraction of diffusing particles relative to all particles (fD) are shown.