Three-dimensional single-particle tracking in live cells: news from the third dimension

A Dupont,M Gorelashvili,D C Lamb,D Arcizet,Y Katayama,F Wehnekamp,D Heinrich,V Schüller

doi:10.1088/1367-2630/15/7/075008

Abstract

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells’ cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.

Highlights

Single-particle tracking (SPT) is of growing importance in the biophysical community
To test the validity of the analysis method, 3D Brownian diffusion of particles with a diffusion coefficient of 0.163 μm2 s−1 was simulated and the 3D particle trajectories and their 2D projections were evaluated by a global mean square displacement (MSD) analysis and by the local MSD algorithm TRAnSpORT [18]
To obtain reliable error bars in the global MSD analysis, the trajectory was cut into 15 segments, which were individually analyzed by fitting their MSD to obtain the α-exponent and the diffusion coefficient

Summary

Introduction

Single-particle tracking (SPT) is of growing importance in the biophysical community. Based on a local mean-square-displacement (MSD) analysis, the TRAnSpORT algorithm, modified to perform the analysis in 3D, dissects a trajectory into different transport events identified as active or passive phases The distribution of these phases was calculated with the 2D and the 3D analyses as well as the diffusion coefficients of passive phases and the velocity of active phases. We described a rolling-average algorithm able to reliably separate periods of active and passive motion from 2D trajectories of particles in live cells [18].

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Discussion

Conclusion