Track projections. Gametocyte tracks over time derived from intravital movies were analyzed using the “Cell tracking” and “MTrackJ” ImageJ plugins and ImarisTrack. A total of 500 different gametocytes were tracked for up to 1 min or the duration of their presence in the field of recording.

Velocity. Speed of motion was calculated as the sum of distances between track locations, measured using Volocity 6.3 software (PerkinElmer), and divided by track duration. The presence of parasites per movie lasted for varying amounts of time depending on the tissue and extravascular or intravascular presence. For normalization purposes, all calculations were based on 1 to 20 measurements acquired in 500-ms intervals.

Calculation of time spent in contact with vessels (lag time). To calculate the lag time, or the time during which gametocytes made contact with and arrested, videos acquired over a 5-min time lapse were used. Lag time was identified from the calculation of average speed through time (total arrest being equal to zero), as well as average gametocyte speed in relation to the speed of neighboring circulating RBCs or parasites. Lag was considered a decrease in speed by 50% or more compared to total cell and gametocyte speed.

Calculation of overall proximity to vessels. In Flk1-GFP mice, the vasculature is GFP-tagged. In UBC-GFP mice, the vascular wall in various organs is visible because of the accumulation of GFP. In addition, in C57BL/6 mice, the vasculature was labeled by injection of FITC-conjugated dextran. Proximity of gametocytes to the vessel wall, as well as number of transmigrations, was calculated using the Volocity 6.3 software (PerkinElmer) or Fiji.

Leakage quantification. The images obtained from intravital images of mice injected with FITC-labeled dextran were converted to a false-color scale (rainbow color) based on dextran fluorescent intensity using ImageJ. Then, the blood vessel area and interstitial space were manually circumscribed, and the MFIs of each area were measured every 500 ms. For determination of individual leakage caused by single parasites, a region of interest was generated, and fluorescence changes (MFI) over a 5-min interval were quantified.

Statistical analysis. Data were displayed in box plots or histograms generated using PRISM 6.0 software. Means, medians, interquartile ranges, and SDs were calculated from three to five independent experiments performed in triplicate, using STATA 13.0 software. The P values were calculated using Student’s t test or analysis of variance (ANOVA) in STATA 13.0. Significance between samples is indicated with asterisks as follows: *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

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