Figure 3.

Diffusion coefficient measurement bias estimated from simulations without noise of a fluorescent molecule confined to a cylinder. Colors indicate the set of parameters (t_frame, σ_PSF) (Table S1), and M_C is defined according to Eq. 3B. (A) STICS analysis by Gaussian fitting of simulations that contain in-frame motion (IFM). Significant, nonmonotonically varying bias occurs for fast characteristic motion, M_C > 1. (B) STICS analysis by Gaussian fitting of simulated movies that exclude in-frame motion. The nonmonotonically varying bias from (A) is still present at all values of M_C considered, although the bias due to in-frame motion is removed. (C and D) The results of analyzing the same data as in (A) and (B), but determining the step size distribution variance in the STICS protocol by directly calculating the variance rather than by fitting the correlations to Gaussian functions. The combination of direct variance calculation and removal of in-frame motion in (D) removes the bias completely. To see this figure in color, go online.