Do microscopy imaging frequency and experiment duration impact the analysis of T cell movement?

Abstract

For years we have investigated whether vaccine-induced CD8 T cells in the liver hunt Plasmodium sporozoites (malaria) or if the T cells find infected hepatocytes randomly. Using previous T cell position data collected with intravital microscopy, we performed numerous analyses, giving two main conclusions. Firstly, using a new metric for detecting attraction using the Von Mises-Fisher distribution in 3D and statistical analyses, we concluded that cells move randomly until a cluster of cells forms around a parasite; then some cells begin to move with bias. Secondly, using simulations where we control the amount of attraction and otherwise replicated the movement of real cells, we concluded that cells are able to move with enough attraction to find parasites, but not enough to have statistically detectable attraction in experimental data. We have thus constructed methods to test how cells move and moreover determined the strength and the limitations of our metrics.

New opportunities arose in the past year as we received new position data, which has some critical differences from the older data. The new data has more frequent imaging of every 12--20 seconds (old data: 90--120 seconds) and shorter experiments of 30 minutes in total length (old data: 120 minutes). The different experiment parameters also affect some properties of the recorded data, including the calculated speeds (whose calculations in the old data we showed to be artificially low due to mathematical properties).

We repeated our work using the new data. Repeating the statistical analyses, we found that cells still move randomly until a cluster of cells forms around a parasite, then begin to move with bias. Repeating the simulations, now using parameters replicating the movement of new cells, we found that cells still are able to move with enough attraction to find parasites, but not enough to have statistically detectable attraction using current methodology.

In summary, despite differing parameters such as imaging frequencies and experiment durations, we make the same conclusions about T cell movement. This serves as both a biological result and a commentary on the potential effects of differing experimental parameters.