The Atomic Force Microscopy (AFM) project has expanded with the addition of a second instrument for studies of macromolecules of biomedical importance. The new AFM is contained within a custom-built cryo-chamber to maintain the instrument at a constant temperature between +50 centigrade and ?50 centigrade. Preliminary data imply that this AFM-cryo-chamber combination can be used to estimate thermodynamic parameters of macromolecules and concomitantly topography and phase changes. This study should provide unique physical- chemical data of complex macromolecules.The application of ?blind deconvolution? algorithms to studies of host-parasite interactions is developing slowly. The approach is being used for two specimen types (wide field fluorescent images and transmitted light histologic sections). In both cases, the technique provides clearer, deblurred 3-dimensional images of very thick tissues (e.g. mosquito gut) than could be obtained by conventional confocal microscopy.Quantitative light microscopy is being used to estimate the levels of fluorescent-labeled substances present within malaria infected erythrocytes. One of the hallmarks of a Plasmodium falciparum malaria infection is the appearance of protrusions on the surface of infected erythrocytes. These protrusions or knobs mediate the adherence of infected erythrocytes to the endothelial cells lining blood vessels in the brain resulting in fatal cerebral complications. In 1983, Gruenberg et al. using a scanning electron microscope (SEM) reported that knobs began appearing on infected erythrocytes at about the trophozoite stage and increased in number and decreased in size during parasite maturation. However, it was impossible for them to accurately determine the developmental stage of the parasite in an individual cell by SEM or ascertain the effects of multiple infection on knob production in an infected erythrocyte. We used the AFM to study infected erythrocytes at magnifications and resolutions exceeding SEM, and integrated the AFM with a light microscope to concomitantly obtain epifluorescence images of the same erythrocyte. This procedure allowed us to determine unambiguously the developmental stage of the parasite as well as the number and size of knobs in single, double and triple infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. Surprisingly, this lesion is present even into the late trophozoite stage. Knobs begin to form in the early trophozoite stage of a P. falciparum infection, and the number of knobs/unit host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. However, neither parasite number nor stage influences knob size. These data imply that the synthesis of knob proteins by one parasite does not influence protein synthesis by other parasites in the same multiply infected erythrocyte. Furthermore, knobs never coalesce even in multiply infected erythrocytes. These observations form the basis for continued AFM studies of this medically important problem.There are two major problems in applying the AFM to biomedical research: sample preparation and scanning probe geometry. We have devoted a great deal of time this year to the resolution of these two problems with varying degrees of success. In an attempt to improve probe geometry, we have formed a collaboration with a physics group in Japan which is developing nanotubes as AFM probes. Preliminary results were variable but recent tests indicate we are able to achieve a much higher lateral resolution with nanotube probes compared with conventional silicon nitride probes. Our goal is to develop probes of sufficient length to be useful for cell biological studies and have a tip radius of approximately 3 nm. We have begun studies documenting the movement of live ookinetes on the surface of mosquito midguts in vitro. Midguts are removed from female Aedes aegypti mosquitoes 24 hours after a blood meal and the midgut epithelium is separated from the blood bolus. The live midguts are spread out flat on glass slides and are covered with a drop of an ookinete suspension; a cover glass is then added and the slide chamber sealed to prevent evaporation. The movement of ookinetes is followed by differential interference contrast. Our preliminary observations show that ookinetes settle onto the midgut surface and move about on the epithelium. Real-time recording of ookinetes moving on the midgut surface has also resulted in the observation of ookinetes invading the midgut. This observation is of interest both in terms of changes in the morphology of the invaded insect gut as well as the possible future use of this system to assay rates of invasion and to identify specific promotors or inhibitors of invasion.We intend to study the movement of ookinetes in great detail. We will record the movement of ookinetes on midgut surfaces in three dimensions, measure their rate of movement and compare it to parasite movement on glass slides or slides coated with extracellular matrix proteins such as collagen and fibronectin. These studies should provide a detailed description of ookinete movement. Of particular interest is the formation of constrictions along the length of the parasite that correlate with binding to the midgut surface. Constrictions are not seen when parasites bind to artificial surfaces such as glass or plastic, suggesting that the constrictions result from obstructions present in the microvillar-associated matrix.
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