Fig. 5
Enhanced-throughput imaging and analysis for PE therapeutic screening. Schematic represents automated imaging and data analysis work flow using the Operetta high-content imaging (HCI) system (Perkin Elmer) where four consecutive steps are followed for maximum throughput of liver stage (LS) screening. Step 1: fluorescent channels are selected based on secondary conjugates and imaging parameters are set based on ×20 magnification (0.4 NA). Step 2: LS parasites are identified by high intensity staining patterns of LS-specific antibody (i.e., anti-rUIS4 for P. vivax or anti-GAPDH for P. falciparum) denoted by line plot profile (1, 2) Step 3: LS parasites are further characterized into size categories by area and roundness to ensure software is correctly selecting the entire parasite. Step 4: After LS parasites are quantified, raw data is imported into CDD vault or Graphpad Prism where % inhibition and dose-response curves are calculated based on total parasites and size. P. vivax LS assays specifically determine compound activity on hypnozoite population. White scale bars represent 5 µm
