Live Parasite Microscopy Examination

Viewing chambers for live parasite microscopic examination were constructed as previously described (7 (link)). All images were recorded on a Nikon Eclipse Ni light microscope fitted with a Hamamatsu C11440 digital camera and Nikon N Plan Apo λ 63×/1.45NA oil immersion objective. For time-lapse video microscopy, differential interference contrast (DIC) images were taken at 10 s intervals over 30 min while fluorescence (mNeon Green) images were taken every 2 min to prevent bleaching. Time-lapse videos were analyzed and annotated using Fiji (44 (link)). For viability staining using the vital mitochondrial dye MitoTracker Red CMXRos (ThermoFisher Scientific; stored as a 10 μM stock in DMSO), the dye was added (20 nM final concentration) to a suspension of schizonts pretreated for 1 h with either DMSO (control, 1% vol/vol) or compound 3j (10 μM). The schizonts were incubated with the dye for 15 min at 37 °C, then washed twice, transferred to a viewing chamber, and observed immediately by dual DIC/fluorescence microscopy.