title: An automated live imaging platform for studying merozoite egress-invasion in malaria cultures.
abstract: Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite-red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.
authors.affiliation: Cavendish Laboratory, University of Cambridge, Cambridge, UK.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Crick
authors.forename: Teresa
authors.initials: T
authors.lastname: Tiffert
authors.forename: Sheel M
authors.initials: SM
authors.lastname: Shah
authors.forename: Jurij
authors.initials: J
authors.lastname: Kotar
authors.forename: Virgilio L
authors.initials: VL
authors.lastname: Lew
authors.forename: Pietro
authors.initials: P
authors.lastname: Cicuta
mesh_terms: D057205:Automation, Laboratory / Q000379:methods; D002460:Cell Line; D004912:Erythrocytes / Q000469:parasitology*; D006790:Host-Parasite Interactions; D006801:Humans; D007091:Image Processing, Computer-Assisted; D052940:Merozoites / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D010963:Plasmodium falciparum / Q000502:physiology*