An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures
An Automated Live Imaging Platform for Studying Merozoite Egress-Invasion in Malaria Cultures
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DOI：
10.1016/j.bpj.2013.01.018
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发表时间：
2013-03-05
影响因子：
3.4
通讯作者：
Cicuta, Pietro
中科院分区：
文献类型：
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作者：
Crick, Alex J.;Tiffert, Teresa;Cicuta, Pietro
大多数严重和致命的疟疾病例是由恶性疟原虫在红细胞内的无性繁殖周期引起的。在这个周期中，最耐人寻味也是最不为人所知的阶段之一是短暂的入侵前阶段，在这段时间里，裂殖子与红细胞的动态相互作用使裂殖子顶端对齐，为穿透做准备。对这一过程中涉及的分子机制的研究面临着艰巨的技术挑战，需要在受控的实验条件下，使用高分辨率显微镜和各种荧光成像工具对活培养物中的裂殖子出口入侵序列进行多次观察。在这里，我们描述了开发全自动、机器人成像平台以实现此类研究的成功的第一步。在具有软件控制电动功能的倒置舞台显微镜上建立了富含裂殖体的恶性疟原虫活培养物。通过应用各种成像过滤器和选择标准，我们识别了可能即将破裂的感染红细胞，并记录了它们的坐标。我们开发了一种视频图像分析来检测和自动记录本研究中记录的40个出口入侵序列中的裂殖子出口事件。我们观察到出口前感染细胞的动态状态有很大的多态，可能反映了导致裂殖子释放的汇合过程的异步性。
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.