N plan apo λ 100x 1.45na oil immersion objective

Time lapse video microscopy of egress was performed as previously described[9 (link)]. Briefly, tightly synchronized schizonts were percoll-enriched and arrested with 1 μM C2 for 4 h. After C2 wash out, DIC and mCherry images were collected every 5 and 25 s, respectively, for 30 min using a Nikon Eclipse Ni-E wide field microscope fitted with a Hamamatsu C11440 digital camera and a Nikon N Plan Apo λ 100x/1.45NA oil immersion objective. For each experiment, videos of the RAP- and DMSO-treated parasites were taken alternately to ensure that possible differences in the rate of egress were not a result of variation in the maturity of the parasite populations. The images were then annotated using Axiovision 3.1 software and exported as AVI movie or TIFF files. Individual egress events were annotated by detailed visual analysis of the movies, and the delay to the time of egress was recorded for each schizont for subsequent statistical analysis. Mean fluorescence intensity values of individual mCherry-expressing schizonts right before and after PVM breakdown were determined from exported raw image files (TIFF format) as described previously[9 (link)] and using the elliptical selection tool and ‘Histogram’ options of ImageJ/Fiji V1.0. DPAP3KO parasites were analyzed to determine the residual background fluorescence derived from the hemozoin.

Segmented schizonts were purified from RAP- or mock-treated PfPDEβ_ΔcatHA cultures as described above, introduced into custom-made viewing chambers [28 (link)], and imaged on a Nikon Eclipse Ni-E widefield microscope with a Hamamatsu C11440 camera and a Nikon N Plan Apo λ 100x/1.45NA oil immersion objective. For egress videos, images were taken at 5-second intervals over a total of 30 minutes. Individual egress events were cropped, trimmed, and converted to video file format in ICY bioimage analysis software. For invasion videos, images were taken every 150 ms for 8 minutes following schizont rupture and processed using the Nikon NIS elements AR software. Merozoites from each rupture event were followed up and scored for their ability to deform the host cell, induce echinocytosis, and complete invasion.