Fig. 1: Resolving molecular diffusion in cytoplasmic extracts.

a Schematic of the experimental setup used in this study: Xenopus laevis eggs were fractionated to obtain the undiluted cytoplasmic extract, which was then deposited on the surface of an imaging dish and covered with mineral oil. b Time-lapse images of the self-organizing cytoplasm, visualized with 1 µM ER-Tracker Red dye. c Image (left) of dilute 100-nm (diameter) microspheres in extracts. Trajectories (right) are examples from single-particle tracking in the organized cytoplasm (interior regions in Fig. 2a). The relative positions between the three tracked particles were adjusted for display (they were tens of microns apart in the extracts). d Ensemble mean squared displacement (MSD) analysis derived from the single-particle tracking. Shaded area, standard error of the mean (SEM). Dashed lines are fitting to \({MSD}\left(t\right)=\Gamma {t}^{\alpha }\), where Γ is the generalized diffusion coefficient³⁴. The fitted {Γ, α} for disorganized and organized cytoplasm are {1.12, 0.75}, and {0.65, 0.78}, respectively. The localization error of these SPT experiments, quantified with immobilized microspheres, was below 0.01 µm² (Supplementary Fig. 1a). e, f Representative FCS data of BSA-Alexa Flour 488 in cytoplasmic extracts. The full intensity trajectory (60 s) is shown in Supplementary Fig. 2c. The inset depicts a molecule diffusing through a confocal spot. G(τ) is the autocorrelation function, where τ is the lag time; \(\delta I\left(t\right)=I\left(t\right)-\left\langle I\left(t\right)\right\rangle\), where I(t) is the fluorescence intensity and the brackets denote time averages. The autocorrelation curve was fitted by an anomalous diffusion model (dashed line). In this example, the number of particles was 42, and the average intensity (shown in e) was 32 kHz, corresponding to a molecular brightness of 0.76 kHz/molecule. Source data are provided as a Source Data file.