Intravital imaging experiments track CD8 T cells searching for Plasmodium liver stages, but do the imaging frequency and duration impact the detection of CD8 T cell bias?

Abstract Vaccine-induced CD8 T cells can provide sterilizing protection by killing Plasmodium sporozoites in mice, but how do they find those parasites? We recently showed that most liver-localized CD8 T cells search for infected hepatocytes randomly, with some cells displaying bias toward the infection site only when there is already a CD8 T cell cluster present. Our initial intravital microscopy experiments minimized tissue damage from laser exposure by using low imaging frequency (volume/1.5–2min) and were fairly lengthy (1–2h). In these experiments, activated CD8 T cells displayed Brownian-like turns and slow average calculated speeds of 1–3 um/min. Recently, we performed new experiments in which we recorded the movement of CD8 T cells at a higher frequency (volume/0.2min) at the cost of shorter experiments (30min). Interestingly, observed speeds ranged between 5 and 18 um/min, suggesting that the calculated speed of CD8 T cells depends on the details of the experiment. Further testing found that imaging frequency impacts the estimated average speed per cell: reducing the imaging frequency from 0.2min to 2min can reduce the average calculated cell speed 5-fold, partially explaining the discrepancy between our datasets’ speeds. Nonetheless, the new data still suggest that some CD8 T cells display attraction towards the infection site only when there is a cluster of CD8 T cells already present. Taken together, our results highlight the impact of details of intravital imaging experiments on inferred parameters of T cell movement in complex environments.