Defining the quantitative limits of intravital two-photon lymphocyte tracking

Abstract

Two-photon microscopy has substantially advanced our understanding of cellular dynamics in the immune system. Cell migration can now be imaged in real time in the living animal. Strikingly, the migration of naive lymphocytes in secondary lymphoid tissue appears predominantly random. It is unclear, however, whether directed migration may escape detection in this random background. Using a combination of mathematical modeling and experimental data, we investigate the extent to which modern two-photon imaging can rule out biologically relevant directed migration. For naive T cells migrating in uninfected lymph nodes (LNs) at average 3D speeds of around 18 μm/min, we rule out uniform directed migration of more than 1.7 μm/min at the 95% confidence level, confirming that T cell migration is indeed mostly random on a timescale of minutes. To investigate whether this finding still holds for longer timescales, we use a 3D simulation of the naive T cell LN transit. A pure random walk predicts a transit time of around 16 h, which is in good agreement with experimental results. A directional bias of only 0.5 μm/min-less than 3% of the cell speed-would already accelerate the transit twofold. These results jointly strengthen the random walk analogy for naive T cell migration in LNs, but they also emphasize that very small deviations from random migration can still be important. Our methods are applicable to cells of any type and can be used to reanalyze existing datasets.lymphocyte migration | statistical analysis | lymph node transit T wo-photon microscopy has fundamentally changed our view of immune cell migration. The first groups who imaged lymphocyte migration in intact organs (1, 2) reported that cells move in a run and tumble fashion and found no evidence for synchronization or directionality. This finding came as a surprise to many immunologists; previous research had emphasized the role of chemokines, "immunology's high impact factors" (3), and therefore, put forward a view of lymphocyte migration being nicely orchestrated. However, lack of evidence for cell synchronization does not necessarily rule out directed migration. Biased deviation from random motion can create an effect that, like directed migration, causes cells to displace gradually. In biology, such deviation is most often caused by an external stimulus, like a chemokine gradient or a flowing liquid, and is then called taxis. Specific ways in which cells could respond to a directed stimulus include faster migration (orthotaxis), preferential turning (topotaxis), and increased persistence (klinotaxis) to the stimulus (Fig. S1). These different kinds of taxis are called taxis modes (4, 5).Strong taxis, which was found in B cells (6) and neutrophils (7), is obvious to the naked eye. However, detecting more subtle taxis can require sophisticated data analysis; for instance, Castellino et al. (8) used angle analysis to show that naive CD8 + T cells move to sites where naive CD4 + T cells and dendritic cells interact. These examples emphasize that detecting...

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