Figure 2.

Precision in measuring the molecular MSD by correlation spectroscopy. (A) ${\mathit{\sigma }}_{\mathit{iMSD}}$, as defined in Supporting Materials and Methods, is quantified for a defined range of molecular brightness (N_ph, number of photons per molecule per frame) and molecular density (N_mol, number of molecules per PSF) values. In detail, the measured ${\mathit{\sigma }}_{\mathit{iMSD}}$ is plotted against N_ph for the two selected brightness values of 0.1 and 10 molecules per PSF (open and solid dots, respectively). The red lines underline the dependence of ${\mathit{\sigma }}_{\mathit{iMSD}}$ on the square root of N_ph. In the inset, a surface plot for all tested conditions is shown. (B) The same plot as in Fig. 1A shows the contribution of spatiotemporal fluctuation analysis to the calculation of ${\mathit{\sigma }}_{\mathit{iMSD}}$ as obtained from simulated experiments for three representative molecular brightness levels of 10, 100, and 1000 kPhs/s (dark blue to cyan solid lines).