Analysing immune cell migration

Key Points To analyse time-lapse video microscopy experiments of the immune system, quantification of cell migration is required. A toolbox of quantitative measures, such as cell speed, motility coefficient, confinement ratio and various angles of migration is available for that purpose. Time-lapse imaging of the immune system is associated with various artefacts than can affect the estimated cell positions over time. As a result, measures of cell migration are also affected by such artefacts, and this may obscure biologically relevant differences between experimental settings or generate spurious results. Imaging artefacts are related either to cell tracking (switching or splitting of tracks, double tracking of cells and errors of tracking near borders) or to imaging itself (imprecise calibration of the axial dimension and small tissue drift). Detection and correction for artefacts can be done using various migration angles. For example, plotting the average angle to the axial border plane versus the distance to that border can help to detect border tracking errors, calibration errors in the axial dimension and small tissue drift. Cell-based measurements are better at detecting distinct subpopulations among cells than step-based approaches. However, an important disadvantage of cell-based versus step-based parameters is that the shape of the imaged space affects the results, and this makes comparison between experiments problematic. Determining contact times between cells in imaging experiments is non-trivial because observed (underestimated) rather than exact contact time is known for most contacts. However, the true contact time distribution can be estimated using a mathematical approach. Time-lapse video microscopy is an important experimental tool for examining the dynamic behaviour of immune cells in situ and involves quantitative analysis of cell migration and cell–cell interactions. Here, several commonly measured parameters and imaging artefacts are described and potential solutions to detect and correct these artefacts are proposed. The visualization of the dynamic behaviour of and interactions between immune cells using time-lapse video microscopy has an important role in modern immunology. To draw robust conclusions, quantification of such cell migration is required. However, imaging experiments are associated with various artefacts that can affect the estimated positions of the immune cells under analysis, which form the basis of any subsequen