In war, it is important to know your enemy. The same is true when fighting disease, especially a deadly one that has grown resistant to drugs. Researchers at Georgetown University in Washington have developed a customized spinning disk confocal microscope that enables them to image — with enough resolution for measurement of features — live malarial parasites wiggling inside human red blood cells. This information could provide a valuable tool in combating malaria. “This helps us understand drug resistance and aids design of new drugs and other therapies,” said Paul D. Roepe, leader of the research team. Roepe, professor of chemistry and of biochemistry, cellular and molecular biology, is co-director of the university’s Center for Infectious Disease. He noted that drug-resistant malaria kills about a million people a year worldwide. Thus, there is tremendous interest in learning more about how antimalarial drugs work so that new drugs can be developed. The nature of malaria makes obtaining this information a challenge. The disease, widespread in the tropics, debilitates millions of people annually worldwide. It is caused by parasites that are mostly transmitted by mosquitoes. The parasites invade blood cells, eventually consuming them and causing fever, anemia and even death. Molecular investigational methods require the use of low-density human blood cultures, which are time-consuming and expensive to produce. So researchers are developing new techniques, including optical ones. Unfortunately, imaging the parasites is challenging because they move inside their host cells, resulting in blurred images when acquired at high resolution via conventional laser scanning confocal microscopy. The parasites also have features of interest that are smaller still. Researchers need the resolution offered by scanning confocal microscopy but at much faster acquisition times. The group, therefore, turned to spinning disk confocal microscopy, which is similar to scanning confocal microscopy in that it uses a pinhole effect to reject most out-of-focus light. However, instead of one pinhole, it uses thousands that are on a disk. A second disk bears microlenses that align with the pinholes on the first disk. The disks spin — synchronized — resulting in a significant improvement in image-acquisition speed. Roepe noted that this technique allows three-dimensional data to be acquired in less than one second, thus eliminating blurring. That, in turn, improves the ability to reliably quantify certain parameters, such as the volume of intracellular compartments. As described in two papers in the Oct. 17 issue of Biochemistry, the researchers modified a commercial spinning disk confocal microscope from PerkinElmer of Wellesley, Mass., adding a cooled CCD camera from Hamamatsu Corp. of Bridgewater, N. J., an argon-krypton laser from Coherent Inc. of Santa Clara, Calif., and, for fast illumination control, an acousto-optic tunable filter from Melbourne, Fla.-based Neos Technologies. They added Nikon differential interference contrast optics, which necessitated a change in the microscope’s stage to one from Merzhauser. Finally, they used a piezoelectric controller from Cambridge, Mass.-based Piezo Systems for fast Z-axis movement of the objective. With this setup, they achieved better than 250-nm resolution in all three dimensions while completing a full scan in less than 800 ms. Other aspects of the technology were algorithms and a customized dual-processor computer to deblur images and to correct for axial smearing, thereby improving the quality of the data. Processing the image files was computationally intensive, with some of the experiments requiring the handling of hundreds of files and 10 GB of raw data. Roepe said that the size of the files and the speed with which they were processed was unusual for a microscopy-based study. While the technology made the imaging possible, the application of cell biology techniques ensured that there was something informative to image. In their investigations, the researchers looked at the development of the parasites under specific conditions and with particular attributes. To gather meaningful data, they had to be able to look at more than one parasite at any given time. “The ability to closely synchronize the parasite cell culture so that every parasite in the culture is at the same stage of development is also important,” Roepe said. They used the techniques and the imaging technology for several studies of malaria. In one, they quantified the development of hemozoin, the crystallization of heme released upon hemoglobin digestion in the parasite, for both drug-sensitive and drug-resistant malaria. They analyzed the growth of hemozoin in the presence and the absence of chloroquine, one of a family of drugs that has been the chief line of defense against malaria. Data from other studies had suggested that such drugs act by inhibiting hemozoin growth in some fashion, but the exact nature of their effects in vivo was not known. These high-resolution differential interference contrast images of hemozoin — the crystallization of heme released upon hemoglobin digestion in the malaria parasite — were taken at various stages of development of the parasite. They were taken in less than 100 ms using spinning disk confocal microscopy. The researchers used chloroquine in doses large enough to affect the parasites that were sensitive and tracked the rate of formation of hemozoin over time. Because hemozoin is optically dense, they could use differential interference contrast, allowing them to use just a few cells in each experiment — an advantage given the difficulty of culturing the parasites in bulk. The measured hemozoin production tracked known visual criteria, with rapid increases in the beginning and a plateau later. The researchers found, though, that, in the presence of chloroquine, the production did not drop until 50 percent of the hemozoin had formed. From that, and the effect of verapamil, an agent known to reverse chloroquine resistance, they concluded that the drug’s toxicity was caused by the buildup of the noncrystalline form of the digestive byproduct. That result agreed with the leading hypothesis about how chloroquine works. However, the different strains produced hemozoin at the same rate and suffered from roughly the same decrease when exposed to chloroquine — an unforeseen result, according to Roepe. “We initially expected to see some difference in the rate of hemozoin production for the drug-resistant versus the drug-sensitive strains of malarial parasites,” he said. In their second study, the scientists looked at chemical and volume differences in the digestive vacuole in drug-resistant malaria, comparing them with the drug-sensitive variety. For the chemistry, they measured the pH of the two strains and found that the digestive vacuoles of the drug-resistant variety are more acidic. For volume measurements, they used fluorescence imaging, with 488-nm excitation from the laser and the fluorophore Oregon Green coupled to dextran internalized in the parasite. They calculated the volume of the vacuole by optically stepping through it in 0.2-μm Z-axis increments and by processing the acquired data, doing this for 20 or more cells for each strain. They found that, at maturity, the drug-resistant strains had larger digestive vacuole volumes than those of the drug-sensitive strains by a factor of about two. One implication of this, Roepe noted, is that the drug concentration in this subcellular compartment would be different for resistant malaria, which could be important in the drug resistance itself. In this fluorescence-based image, changes in the volume of the digestive vacuole of the parasite responsible for malaria are quantified at different times before removal of chloride from the perfusate flowing over the cells (top), during removal (middle) and after chloride replacement (bottom). Images courtesy of Paul D. Roepe, Georgetown University. The researchers are making hemozoin-formation measurements using different drugs and strains, experimenting with various fluorescent labels, and developing methods to simultaneously monitor dual- and triple-labeled cells. On the equipment side, they are developing ratiometric spinning disk confocal microscopy imaging as well as the ability to acquire differential interference contrast and fluorescence data at the same resolution from the same cell. “We should be making fairly substantial progress on all these fronts over the next year or so,” Roepe said.