Viewing chambers were constructed as previously described (41 (link)). Images were recorded on a Nikon Eclipse Ni light microscope fitted with a Hamamatsu C11440 digital camera and Nikon N Plan Apo λ 63×/1.45 NA oil immersion objective. For time-lapse video microscopy, vehicle (DMSO) or RAP-treated schizonts were synchronized by incubation with the reversible PKG inhibitor compound 2 and then washed, mixed in equal proportions, and monitored for egress. Differential inference contrast (DIC) images were taken at 10-s intervals over 45 min, while fluorescence (GFP or mCherry) images were taken every 5 to 10 min to prevent bleaching. Time-lapse videos were analyzed and annotated using Fiji (57 (link)).
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Time-lapse Microscopy of Malaria Parasite Egress
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Corresponding Organization :
Other organizations : University of Geneva, The Francis Crick Institute, GlaxoSmithKline (Germany), GlaxoSmithKline (Spain), University of London
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Variable analysis
independent variables
- Treatment with RAP (vehicle DMSO or RAP)
- Egress of schizonts
- Synchronized schizonts by incubation with the reversible PKG inhibitor compound 2
- Positive control: DMSO-treated schizonts
- Negative control: RAP-treated schizonts
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