Gligorijevic Bojana, McAllister Ryan, Urbach Jeffrey S, Roepe Paul D
Department of Chemistry, Program in Tumor Biology, Lombardi Cancer Center, Georgetown University, 37 and O Streets, Washington, DC 20057, USA.
Biochemistry. 2006 Oct 17;45(41):12400-10. doi: 10.1021/bi061033f.
We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.
我们定制了一台尼普科夫旋转盘共聚焦显微镜（SDCM），用于获取活的红细胞内疟原虫的三维（3D）随时间变化的数据。由于活的疟原虫在红细胞内蠕动，传统的激光扫描共聚焦显微镜在重建z轴堆叠数据后会产生模糊的3D图像。相比之下，由于SDCM能够在数百毫秒内获取高x、y和z分辨率的数据集，因此活疟原虫细胞生物化学的关键方面在生理意义上的时间尺度上能够得到更好的解析。在本文中，我们展示了活疟原虫的首张3D微分干涉差（DIC）透射率“z轴堆叠”图像，并利用这些图像在生理条件下对活疟原虫消化泡内产生的疟色素（Hz）进行定量分析。通过使用活的同步培养物以及对锐化后的DIC z轴堆叠进行体素分析，我们首次对氯喹敏感（CQS）和耐药（CQR）疟原虫的Hz生长速率进行了体内定量分析。我们展示了实验室菌株以及表达赋予CQR特性的突变型pfcrt基因的pfcrt转染体的数据。我们还分析了在存在和不存在生理相关剂量的氯喹（CQ）和维拉帕米（VPL）的情况下Hz的生长速率，从而首次对著名的CQ药理学菲奇假说的关键预测进行了体内定量。在接下来的论文[Gligorijevic, B.,等人（2006年）《生物化学》45卷，第12411 - 12423页]中，我们通过SDCM获取活疟原虫消化泡的荧光图像，并利用这些图像对CQS和CQR疟原虫的消化泡体积进行定量分析。
