Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes’ Polydispersity

Abstract

This work, based on in vivo and in vitro measurements, as well as in silico simulations, provides a consistent analysis of diffusion of polydisperse nanoparticles in the cytoplasm of living cells. Using the example of fluorescence correlation spectroscopy (FCS), we show the effect of polydispersity of probes on the experimental results. Although individual probes undergo normal diffusion, in the ensemble of probes, an effective broadening of the distribution of diffusion times occurs-similar to anomalous diffusion. We introduced fluorescently labeled dextrans into the cytoplasm of HeLa cells and found that cytoplasmic hydrodynamic drag, exponentially dependent on probe size, extraordinarily broadens the distribution of diffusion times across the focal volume. As a result, the in vivo FCS data were effectively fitted with the anomalous subdiffusion model while for a monodisperse probe the normal diffusion model was most suitable. Diffusion time obtained from the anomalous diffusion model corresponds to a probe whose size is determined by the weight-average molecular weight of the polymer. The apparent anomaly exponent decreases with increasing polydispersity of the probes. Our results and methodology can be applied in intracellular studies of the mobility of nanoparticles, polymers, or oligomerizing proteins.

Determination of oligomerization state of Drp1 protein in living cells at nanomolar concentrations

“…Average amplitudes of dimer and tetramer in cytosol were 0.34 and 0.15 respectively. In-depth analysis of the amplitudes of FCS autocorrelation curves led to a conclusion that all oligomers detected by FCS exhibit the same brightness, which is equal to one EGFP molecule (our reasoning is presented in SI8) 36–38 . It follows that FCS amplitudes correspond to actual concentrations and that the average relative quantity of dimers to tetramers in cytosol is 7:3.…”

“…Briefly, a two component model (as in case of Drp1 mutants) resulted in a poor fit and the value of diffusion time was in-between those expected for the dimer and tetramer. This suggested that there is a mixture of dimer and tetramer in the cytosol (see SI7) 36 . Therefore, fitting was performed using three-component model with diffusion times fixed as values expected for dimer (9.1 ± 0.5 µm 2 /s) and tetramer (5.7 ± 0.4 µm 2 /s), derived from Eq.…”

“…Fluorescent signal for FCS was collected by Single Photon Avalanche Diodes (MPD and PerkinElmer). Each experiment was preceded by a calibration using Rhodamine B (λ ex = 543 nm, Sigma-Aldrich) dissolved in a solution of 2.5% wt glucose in PBS 36 . The objective correction collar was adjusted to provide optimal FCS read-out, and RhoB diffusion was used for precise characterization of focal volume size and shape.…”

Single quantum dot tracking reveals the impact of nanoparticle surface on intracellular state

“…Doing so would require >10 minutes of data acquisition that would be highly phototoxic to cells. FCS in general can be challenging to interpret as polydispersity and anomalous diffusion cannot be independently distinguished in time-traces 52 , and single-QD intermittency can interfere with time-correlation analyses 53 . The ability of single-particle techniques to provide direct insight into particle heterogeneity is thus a major benefit, compared with ensemble techniques.…”

Transport of nanoprobes in multicellular spheroids

“…The experiments were preceded by establishing the dimension of the confocal volume using rhodamine B (Sigma-Aldrich, USA) dissolved in 2.5% glucose in PBS. 18 FCS measurements were performed at a depth of 10-30 µm within the spheroids (Fig. 1), in the extracellular space at the distance of 2-10 µm from the cell edges.…”