Abstract
Darkfield and confocal laser scanning microscopy both allow for a simultaneous observation of live cells and single nanoparticles. Accordingly, a characterization of nanoparticle uptake and intracellular mobility appears possible within living cells. Single particle tracking allows to measure the size of a diffusing particle close to a cell. However, within the more complex system of a cell’s cytoplasm normal, confined or anomalous diffusion together with directed motion may occur. In this work we present a method to automatically classify and segment single trajectories into their respective motion types. Single trajectories were found to contain more than one motion type. We have trained a random forest with 9 different features. The average error over all motion types for synthetic trajectories was 7.2%. The software was successfully applied to trajectories of positive controls for normal- and constrained diffusion. Trajectories captured by nanoparticle tracking analysis served as positive control for normal diffusion. Nanoparticles inserted into a diblock copolymer membrane was used to generate constrained diffusion. Finally we segmented trajectories of diffusing (nano-)particles in V79 cells captured with both darkfield- and confocal laser scanning microscopy. The software called “TraJClassifier” is freely available as ImageJ/Fiji plugin via https://git.io/v6uz2.
Highlights
Transport processes of particulate structures inside cells are of pivotal importance for many cellular functions
We have reported first results obtained with a new method which classifies normal diffusion, subdiffusion and directed motion using a random forests approach trained by three features which were estimated for simulated trajectories [12]
The trained model will be applied to experimental trajectories from cell and other studies to demonstrate the usefulness of the method
Summary
Transport processes of particulate structures inside cells are of pivotal importance for many cellular functions. The way how small objects move at the cell boundary may provide insight into mechanical properties of the local surroundings [1], and can unravel nanoparticle (NP) or even protein cell entry mechanisms [2,3,4]. In all these cases, single objects need to be imaged and their trajectories carefully analyzed. Particle movements can be classified into four basic motion types: normal diffusion (ND), anomalous diffusion (AD), confined diffusion (CD) or directed motion (DM). PLOS ONE | DOI:10.1371/journal.pone.0170165 January 20, 2017
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