Humic substances (HSs) are the natural organic polyelectrolytes formed from the biochemical weathering of plant and animal remains. Their macromolecular structure and chemistry determine their role in biogeochemical processes. In situ spectromicroscopic evidence showed that the HS macromolecular structures (size and shape) vary as a function of HS origin (soil versus fluvial), solution chemistry, and the associated mineralogy. The HSs do not simply form coils in acidic or strong electrolyte solutions and elongated structures in dilute alkaline solutions. The macromolecular structural changes of HSs are likely to modify contaminant solubility, biotransformation, and the carbon cycle in soils and sediments.

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 microm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive "water window", where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.

Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitortreated, infected erythrocytes, high concentrations of material were detected in abnormal digestive vacuoles and aggregated at the parasite plasma membrane. We have demonstrated that an abnormal host erythrocyte skeleton affects structural development of parasites and that this aberrant development can be detected in the following generation when parasites from protein 4.1-deficient red blood cells infect normal erythrocytes. This work extends our current understanding of the relationship between the host erythrocyte membrane and the intraerythrocytic malaria parasite by demonstrating for the first time that constituents of the erythrocyte membrane play a role in normal parasite structural development.

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Interfaces between minerals and water, and minerals and microbes, are chemically complex and have traditionally been considered beyond the capabilities of surface science techniques, except for model systems under controlled laboratory conditions. We report on some advances in soft xray spectroscopy and imaging that make it possible to extract meaningful chemical information about interfaces of specimens that have complex histories, involving environmental exposure. These measurements utilize x-ray absorption spectroscopy, in combination with spatial resolution, in a technique called x-ray spectro-microscopy. Examples are drawn from attempts at Mn and Fe speciation of biologically produced minerals, bio-corrosion deposits, and clays.

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