Figure 2.

Diffusion coefficient measurement bias estimated from simulations of unconfined diffusion of a fluorescent molecule with no noise. Colors indicate the set of parameters (t_frame, σ_PSF) (Table S1), and M_C is defined according to Eq. 3A. (A) STICS analysis by Gaussian fitting of simulations that contain in-frame motion (IFM). Significant bias is measured for M_C > 1. (B) STICS analysis by Gaussian fitting of simulated movies that exclude in-frame motion. (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. Both the removal of in-frame motion in (B) and the direct calculation of variance in (C) and (D) eliminate the measurement bias. To see this figure in color, go online.