GFP fluorescent P. falciparum and changes in fluorescence over the course of the parasite asexual lifecycle. (A) Trophozoite stage PfM3' parasite transfected with the pHGBrHrBl-1/2 plasmid expressing GFP. Selected images show GFP fluorescence, DAPI staining of the nucleus, and bright field image showing the infected erythrocyte. Merged images demonstrate GFP fluorescence appears largely restricted to the parasite cytosol. (B) Changes in GFP mean fluorescence intensity of tightly synchronized PfPHG parasites followed through 48 hours of the parasite lifecycle by flow cytometry (detected in channel Fl-1). (C) Changes in EtBr mean fluorescent intensity (detected in channel FL-2) of the same parasites stained with 10 μg/ml EtBr followed through 48 hours of the parasite lifecycle. Detection of early ring stage parasites by flow cytometry using (D) GFP fluorescence or (E) EtBr fluorescence highlighting the poor separation commonly seen between EtBr stained rings and uninfected RBCs.