Fig. 3

The positional uncertainty in 2D and 3D simulations depends on the brightness and the temporal resolution. a The localization uncertainty improves with increasing number of photons emitted from the GNR. The blue line represents the theoretical value obtained from Eq. 1. The images obtained from simulations were analyzed in 2D (fitting only one slice per 3D stack, blue dots) and in 3D (fitting the whole 3D stack, black squares). In 3D, the localization accuracy increases as compared to the 2D case. Each point in the graph is an average of 10 sets of 200 simulated images. The background noise was kept constant at b = 0.5. b The effect of the movement of the particle between frames for an acquisition time of 10 fr/s. As expected, the uncertainty increased with the diffusion coefficient