High resolution protein localization using soft X-ray microscopy.

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.

Automatic image acquisition, calibration and montage assembly for biological X-ray microscopy.

We describe a system for the automatic acquisition and processing of digital images in a high-resolution X-ray microscope, including the formation of large-field high-resolution image montages. A computer-controlled sample positioning stage provides approximate coordinates for each high-resolution subimage. Individual subimages are corrected to compensate for time-varying, non-uniform illumination and CCD-related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large-field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X-ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X-ray microscopical study to date.

High resolution transmission soft X-ray microscopy of deterioration products developed in large concrete dams

In concrete structures, the reaction of certain siliceous aggregates with the highly alkaline concrete pore solution produces an alkali-silicate gel that can absorb water and expand. This reaction can lead to expansion, cracking, increased permeability, and decreased strength of the concrete. Massive concrete structures, such as dams, are particularly susceptible to the damage caused by the alkali-silica reaction because of the availability of water and because massive gravity dams usually do not contain steel reinforcement to restrain the expansion. Both the cement hydration products and alkali-silica reaction products are extremely sensitive to humidity. Consequently, characterization techniques that require high vacuum or drying, as many existing techniques do, are not particularly appropriate for the study of the alkali-silica reaction because artefacts are introduced. Environmental scanning electron micrographs and scanning electron micrographs with energy dispersive X-ray analysis results demonstrate the effect of drying on the morphology and chemical composition of the alkali-silicate reaction gel. Thus, the impetus for this research was the need to observe and characterize the alkali-silica reaction and its gel product on a microscopic level in a wet environment (i.e. without introducing artefacts due to drying). Only soft X-ray transmission microscopy provides the required high spatial resolution needed to observe the reaction process in situ. The alkali-silica reaction can be observed over time, in a wet condition, and at normal pressures, features unavailable with most other high resolution techniques. Soft X-rays also reveal information on the internal structure of the sample. The purpose of this paper is to present research, obtained using transmission soft X-ray microscopy, on the effect of concrete pore solution cations, namely sodium and calcium, on the product formed as a result of alkali attack. Alkali-silicate reaction (ASR) gel was obtained from the FURNAS Dam in Minas Gerais, Brazil. Images of the ASR gel in sodium hydroxide indicated dissolution and repolymerization of the silicate into a less dense form, demonstrating the expansive nature of the gel when exposed to alkalis. In the calcium hydroxide solution, ASR gel, silica fume, and chemical grade silica gel each reacted with the calcium ions in solution to produce a calcium silicate hydrate precursor with a lathlike, branching morphology. The distinctive spherulitic microstructure formed during this reaction was identified as the 'sheaf of wheat' morphology, previously described in the literature. In addition, the development of the sheaf of wheat morphology was documented over time. These results suggest that of the cations studied in this investigation, it is the alkalis in concrete pore solution that produce the expansive ASR gel, while reaction with calcium ions does not result in expansion or damage to the concrete structure. More broadly, these results demonstrate the advantage of transmission soft X-ray microscopy for the study of the alkali-silica reaction, indicating the value of this technique for further studies in concrete technology.

Imaging of humic substance macromolecular structures in water and soils

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.

X-ray microscopic visualization of specific labeling of adhesive molecule CD36 and cytoadherence by Plasmodium falciparum infected erythrocytes.

We investigated the cytoadherence of Plasmodium falciparum infected erythrocytes to target cells that express CD36 by soft x-ray microscopy. Using immunogold beads enhanced with silver, we localized CD36 on the surface of intact melanoma cells and throughout Triton extracted melanoma cells. We examined the orientation of parasites within erythrocytes that bound to target cells, and the interactions between the red cell membrane and the target cell, and we confirmed that fibrillar structures on the surface of melanoma and endothelial cells can be involved in the association between infected erythrocytes and melanoma cells or endothelial cells.

Resolution determination in X-ray microscopy: an analysis of the effects of partial coherence and illumination spectrum.

Determining the resolution of a zone plate X-ray microscope is a complicated issue, depending on many factors in addition to the quality of the optic. These include the degree of coherence of the illumination, the illumination spectrum, and the nature of the resolution test object. In this paper, we examine closely how the degree of coherence of the illumination affects the resolution as measured with three typical test patterns. In addition, we determine the extent to which the illumination spectrum affects the resolution. We compare X-ray microscope test images to numerical simulations as a function of coherence and illumination spectrum. We are able to conclude that in these experiments, the resolution of the X-ray microscope is 41 nm, or 40% discrepancy is due to aberrations in the objective zone plate lens.

Intracellular structures of normal and aberrant Plasmodium falciparum malaria parasites imaged by soft x-ray microscopy.

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 inhibitor-treated, 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.

Partially coherent image formation with x-ray microscopes.

Image formation with partially coherent radiation is evaluated with the Hopkins formula and then applied to x-ray microscopy. Image characteristics expected from instruments with circular and annular pupils in partially coherent conditions are considered for two-point objects and a knife-edge object. The theoretically expected values for image characteristics that are easy accessible by an experiment, such as the width of a knife edge, are given for various x-ray microscopes.

X ray microscopy--state of the art and expected developments.

The Göttingen X ray microscope at the electron storage ring BESSY in Berlin and X ray microscopy experiments with biological specimens are described. A look ahead to future developments--optics for higher resolution and with better efficiency, detector devices with higher detective quantum efficiency, and the development of a laboratory X ray microscope with a plasma X ray source--is taken.