The morphology of micrometer-sized beta-hematin crystals (synthetic malaria pigment) was determined by TEM images and diffraction, and by grazing incidence synchrotron X-ray diffraction at the air-water interface. The needle-like crystals are bounded by sharp {100} and {010} side faces, and capped by {011} and, to a lesser extent, by {001} end faces, in agreement with hemozoin (malaria pigment) crystals. The beta-hematin crystals grown in the presence of 10% chloroquine or quinine took appreciably longer to precipitate and tended to be symmetrically tapered toward both ends of the needle, due to stereoselective additive binding to {001} or {011} ledges. Evidence, but marginal, is presented that additives reduce crystal mosaic domain size along the needle axis, based on X-ray powder diffraction data. Coherent grazing exit X-ray diffraction suggests that the mosaic domains are smaller and less structurally stable than in pure crystals. IR-ATR and Raman spectra indicate molecular based differences due to a modification of surface and bulk propionic acid groups, following additive binding and a molecular rearrangement in the environment of the bulk sites poisoned by occluded quinoline. These results provided incentive to examine computationally whether hemozoin may be a target of antimalarial drugs diethylamino-alkoxyxanthones and artemisinin. A variation in activity of the former as a function of the alkoxy chain length is correlated with computed binding energy to {001} and {011} faces of beta-hematin. A model is proposed for artemisinin activity involving hemozoin nucleation inhibition via artemisinin-beta-hematin adducts bound to the principal crystal faces. Regarding nucleation of hemozoin inside the digestive vacuole of the malaria parasite, nucleation via the vacuole's membranous surface is proposed, based on a reported hemozoin alignment. As a test, a dibehenoyl-phosphatidylcholine monolayer transferred onto OTS-Si wafer nucleated far more beta-hematin crystals, albeit randomly oriented, than a reference OTS-Si.

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

Synchrotron Fourier transform infrared (FT-IR) spectra of fixed single erythrocytes infected with Plasmodium falciparum at different stages of the intraerythrocytic cycle are presented for the first time. Bands assigned to the hemozoin moiety at 1712, 1664, and 1209 cm(-1) are observed in FT-IR difference spectra between uninfected erythrocytes and infected trophozoites. These bands are also found to be important contributors in separating the trophozoite spectra from the uninfected cell spectra in principal components analysis. All stages of the intraerythrocytic lifecycle of the malarial parasite, including the ring and schizont stage, can be differentiated by visual inspection of the C-H stretching region (3100-2800 cm(-1)) and by using principal components analysis. Bands at 2922, 2852, and 1738 cm(-1) assigned to the nu(asym)(CH(2) acyl chain lipids), nu(sym)(CH(2) acyl chain lipids), and the ester carbonyl band, respectively, increase as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage. Training of an artificial neural network showed that excellent automated spectroscopic discrimination between P. falciparum-infected cells and the control cells is possible. FT-IR difference spectra indicate a change in the production of unsaturated fatty acids as the parasite matures. The ring stage spectrum shows bands associated with cis unsaturated fatty acids. The schizont stage spectrum displays no evidence of cis bands and suggests an increase in saturated fatty acids. These results demonstrate that different phases of the P. falciparum intraerthyrocytic life cycle are characterized by different lipid compositions giving rise to distinct spectral profiles in the C-H stretching region. This insight paves the way for an automated infrared-based technology capable of diagnosing malaria at all intraerythrocytic stages of the parasite's life cycle.