Observation of biological materials by X-ray photoelectron-conversion contact microscopy

Dried biological specimens, such as fossil diatoms, collagen, nerve tissue and spicule of Trepang, were observed by X-ray photoelectron-conversion contact microscopy. A spatial resolution of 0·2 μm was attained. The fossil diatom image shows a clear difference below and above the carbon K-absorption edge (4·46nm).     

Comparative analysis of isolated cellular organelles by means of soft X-ray contact microscopy with laser-plasma source and transmission electron microscopy

Soft X‐ray contact microscopy (SXCM) is, at present, a useful tool for the examination at submicrometre resolution of biological systems maintained in their natural hydrated conditions. Among current X‐ray‐generating devices, laser‐plasma sources are now easily available and, owing to their pulse nature, offer the opportunity to observe living biological samples before radiation damage occurs, even if the resolution achievable is not as high as with synchrotron‐produced X‐rays. To assess the potential of laser‐plasma source SXCM in the study of cellular organelles, we applied it for the analysis of chloroplasts extracted from spinach leaves and mitochondria isolated from bovine heart and liver. X‐ray radiation was generated by a nanosecond laser‐plasma source, produced by a single shot excimer XeCl laser focused onto an yttrium target. The images obtained with SXCM were then compared with those produced by transmission electron microscopy observation of the same samples prepared with negative staining, a technique requiring no chemical fixation, in order to facilitate their interpretation and test the applicability of SXCM imaging.     

Soft X-ray contact imaging of nucleolar chromatin using synchrotron radiation: a comparative scanning and transmission electron microscope study

Contact images (CI) of dehydrated, nucleolar chromatin from amphibian oocytes have been produced by soft X-rays from a synchrotron radiation source. These CI have been compared with the morphology of the original chromatin as seen in scanning and transmission electron microscopes. The quality and informational content of the CI depend very much on certain preparative procedures. The following factors have a marked effect on image quality and need to be carefully controlled: the total X-ray dose, the time and nature of development and the distance of the specimen from the photoresist. The preparation of the chromatin itself, providing that it is critically point dried, is less important. By following a regime of high X-ray dose, sufficient for penetration of the rather thick chromatin rings, and gentle development so that fine detail is not dissolved from the resist surface, it has been possible to obtain images which closely resemble the original chromatin, although the detailed resolution of the CI is not as clear. The smallest biological structures clearly resolved in the CI are ribonucleoprotein granules, which vary in size from 200 to 800 nm. However, by further refinement of preparative conditions it should be possible to improve on the informational content of these images.     

Non-destructive and quantitative imaging of a nano-structured microchip by ptychographic hard X-ray scanning microscopy

We used hard X-ray scanning microscopy with ptychographic coherent diffraction contrast to image a front-end processed passivated microchip fabricated in 80 nm technology. No sample preparation was needed to image buried interconnects and contact layers with a spatial resolution of slightly better than 40 nm. The phase shift in the sample is obtained quantitatively. With the additional knowledge of the elemental composition determined in parallel by X-ray fluorescence mapping, quantitative information about specific nanostructures is obtained. A significant enhancement in signal-to-noise ratio and spatial resolution is achieved compared to conventional hard X-ray scanning microscopy.     

In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy

In this study, we use combined two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and third‐harmonic generation (THG) measurements to image cellular structures of the nematode Caenorhabditis elegans, in vivo. To our knowledge, this is the first time that a THG modality is employed to image live C. elegans specimens. Femtosecond laser pulses (1028 nm) were utilized for excitation. Detailed and specific structural and anatomical features can be visualized, by recording THG signals. Thus, the combination of three image‐contrast modes (TPEF‐SHG‐THG) in a single instrument has the potential to provide unique and complementary information about the structure and function of tissues and individual cells of live biological specimens.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.     

Comparisons of the low-resolution structures of ornithine decarboxylase by electron microscopy and X-ray crystallography: The utility of methylamine tungstate stain and butvar support film in the study of macromolecules by transmission electron microscopy

The structure of ornithine decarboxylase (M_r ≈? 1.04 × 10⁶) from Lactobacillus 30a was investigated by electron microscopy and x-ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod-shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod-shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x-ray crystallographic analysis. A 4.0-Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x-ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.