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Correlation of dual colour single particle trajectories for improved detection and analysis of interactions in living cells.

ABSTRACT: Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.

SUBMITTER: Deschout H

PROVIDER: S-EPMC3759922 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Correlation of dual colour single particle trajectories for improved detection and analysis of interactions in living cells.

Deschout Hendrik H Martens Thomas T Vercauteren Dries D Remaut Katrien K Demeester Jo J De Smedt Stefaan C SC Neyts Kristiaan K Braeckmans Kevin K

International journal of molecular sciences 20130808 8

Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a ...[more]

PMID: 23965965

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Project description:We develop a Bayesian analysis framework to detect heterogeneity in the diffusive behaviour of single particle trajectories on cells, implementing model selection to classify trajectories as either consistent with Brownian motion or with a two-state (diffusion coefficient) switching model. The incorporation of localisation accuracy is essential, as otherwise false detection of switching within a trajectory was observed and diffusion coefficient estimates were inflated. Since our analysis is on a single trajectory basis, we are able to examine heterogeneity between trajectories in a quantitative manner. Applying our method to the lymphocyte function-associated antigen 1 (LFA-1) receptor tagged with latex beads (4 s trajectories at 1000 frames s(-1)), both intra- and inter-trajectory heterogeneity were detected; 12-26% of trajectories display clear switching between diffusive states dependent on condition, whilst the inter-trajectory variability is highly structured with the diffusion coefficients being related by D1 = 0.68D0 - 1.5 × 10(4) nm2 s(-1), suggestive that on these time scales we are detecting switching due to a single process. Further, the inter-trajectory variability of the diffusion coefficient estimates (1.6 × 10(2) - 2.6 × 10(5) nm2 s(-1)) is very much larger than the measurement uncertainty within trajectories, suggesting that LFA-1 aggregation and cytoskeletal interactions are significantly affecting mobility, whilst the timescales of these processes are distinctly different giving rise to inter- and intra-trajectory variability. There is also an 'immobile' state (defined as D < 3.0 × 103 nm2 s-1) that is rarely involved in switching, immobility occurring with the highest frequency (47%) under T cell activation (phorbol-12-myristate-13-acetate (PMA) treatment) with enhanced cytoskeletal attachment (calpain inhibition). Such 'immobile' states frequently display slow linear drift, potentially reflecting binding to a dynamic actin cortex. Our methods allow significantly more information to be extracted from individual trajectories (ultimately limited by time resolution and time-series length), and allow statistical comparisons between trajectories thereby quantifying inter-trajectory heterogeneity. Such methods will be highly informative for the construction and fitting of molecule mobility models within membranes incorporating aggregation, binding to the cytoskeleton, or traversing membrane microdomains.

Dataset Information

Correlation of dual colour single particle trajectories for improved detection and analysis of interactions in living cells.

Publications

Correlation of dual colour single particle trajectories for improved detection and analysis of interactions in living cells.

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