Ultraviolet Germicidal Irradiation and Its Effects on Elemental Distributions in Mouse Embryonic Fibroblast Cells in X-Ray Fluorescence Microanalysis

Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.     

Soft x-ray microscopy

Soft x-ray microscopes are beginning to provide information to complement that obtained from optical and electron microscopy. Soft x-ray microscopy can deliver 30-nm resolution images of hydrated cells up to approximately 10 microns thick, and efforts towards obtaining higher resolution are under way. Although living specimens cannot be studied readily except in single exposures, fixed samples can be imaged at high resolution, and flash-frozen specimens can be studied without chemical modification and without significant radiation damage. Tomography is being developed for 3-D imaging, and spectromicroscopy offers unique capabilities for biochemical mapping of unlabelled structures beyond those of gold and fluorescent labels. Currently, most soft x-ray microscopes operate at synchrotron radiation facilities, but laboratory-scale microscopes are being developed too.     

X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.     

ELECTRON MICROSCOPY AND X-RAY SCANNING MICROANALYSIS OF NEEDLE BIOPSY MATERIAL FROM HUMAN LIVER

A study has been made of the fine structure of hepatic parenchymal cells of human biopsy material in a case of pancreatic tumor with obstructive jaundice. Dense particles about 60 A in diameter have been found in the cytoplasm, which are considered to be ferritin molecules by electron microscopy. They are encountered throughout the cytoplasmic matrix and are often aggregated in electron-transparent areas, most of which are enclosed by an apparently single-layered membrane. Identification of the elemental iron has been pursued by the application of the x-ray scanning microanalyser which reveals a quantitative value within 1.0 per cent of the pure iron sample. The use of x-ray scanning microanalysis enables one to obtain accurate data from extremely small and precisely defined volumes of biological specimens.     

大肠杆菌细胞的软X射线显微成像研究

软X射线显微术是研究含水甚至活性生物样品的有力工具。相对于光学显微镜 ,它具有更高的成象分辨率 ;相对于电子显微镜 ,它的样品制备简单—无须对样品进行脱水、染色和超薄切片等。报道的是利用合肥同步辐射X射线源和接触显微成象技术 ,对自然状态下含水的完整XL1 blueMRF′细菌细胞进行显微成象研究。从获得的显微图象中可以看出一些新的现象。含有DNA、蛋白质的拟核以及中体对波长 2 .4nmX射线具有较弱的吸收能力 ;不少细菌细胞的两端对 2 .4nm波长的X射线的吸收也具有很大的差异。这些有趣现象产生的根本原因和生物学意义有待进一步研究。     

Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.     

Neutron microscopy. The low-damage imaging of specialized organic materials

It is shown that, insofar as radiation damage is concerned, transmission neutron microscopy using neutrons in the energy range approximately 0.0001-1.0 eV is extremely attractive for the imaging of specialized organic materials. By "specialized organic materials" is meant organic specimens composed entirely of specific isotopes that have been selected on the basis of their favorable properties with regard to radiation damage. In connection with such specimens, it is demonstrated that at a resolution of, for example, 100 A, neutrons will have an advantage over soft X-rays in terms of radiation damage, provided that the inherent (neutron) bright field image contrast turns out to be greater than 10(-5). Suggestions relating to (a) the comprehensive calculation of the radiation damage sustained by specialized organic specimens under slow neutron irradiation, (b) the construction of a theory of image formation in the neutron microscope, (c) the development of neutron lenses/focusing devices, and (d) the development of a brighter neutron source (essential for neutron microscopy) are outlined in some detail. The paper concludes with two appendices, which provide important background material.     

X-RAY AND CRYSTALLOGRAPHIC STUDIES OF PLANT VIRUS PREPARATIONS. III

These papers give an account of an optical and x-ray examination of preparations of plant virus substances isolated by Bawden and Pirie, in particular of those of tobacco mosaic disease. They open with a historical survey of the work, indicating the order in which new phenomena were discovered. The subsequent treatment is divided into three parts: I. Introduction and preparation of specimens. II. Modes of aggregation of virus particles. III. (1) The structure of the particles. (2) Biological implications. Part I, after an historical introduction, describes the method of preparation, from solutions of the virus, of optically oriented specimens of different concentrations. For their examination special x-ray apparatus was developed, in particular cameras working with very low angles and capable of indicating spacings up to 1000 Å. In Part III, Section 1 deals with the x-ray evidence on the internal structure of the particles. Even in solution, they have an inner regularity like that of a crystal. Virus preparations are thus in a sense doubly crystalline. Closer analysis reveals that the x-ray patterns are not directly comparable to those of a crystal as many of the reflections do not obey Bragg''s law, but can be understood on the theory of gratings of limited size. The structure seems to consist of sub-units of the dimensions of approximately 11 Å cube, fitted together in a hexagonal or pseudohexagonal lattice of dimensions—a = 87 Å, c = 68 Å. Contrary to what earlier observations seemed to indicate, the particle seems to be virtually unchanged by drying and must therefore contain little water. There are marked resemblances with the structure of both crystalline and fibrous protein, but the virus structure does not belong to any of the classes hitherto studied. There are indications that the inner structure is of a simpler character than that of the molecules of crystalline proteins. Part III, Section 2 contains a comparative study of the optical and x-ray examinations of three strains of tobacco mosaic virus, two of cucumber disease virus, two of potato virus X, and the virus of bushy stunt disease of tomato. In the last case x-ray measurement confirmed the deduction from its cubic crystal habit that it was composed of spherical rather than long particles, and showed that these had a diameter when dry of 276 Å and were arranged in a body-centred cubic close packing. This single example is sufficient to show that the elongated particle form which gives rise to all the anomalous physical properties of the other viruses studied is of no essential biological importance. The similarity and differences observed between the physical properties of these preparations run closely parallel to their clinical and serological classification. Finally, the biological implications of these results are discussed together with possible applications of the new methods of examination to the study of colloid and biological problems.     

Imaging biological structures with the cryo atomic force microscope.

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.     

Analysis of effects of friction on the deformation behavior of soft tissues in unconfined compression tests

Frictionless specimen/platen contact in unconfined compression tests has traditionally been assumed in determining material properties of soft tissues via an analytical solution. In the present study, the suitability of this assumption was examined using a finite element method. The effect of the specimen/platen friction on the mechanical characteristics of soft tissues in unconfined compression was analyzed based on the published experimental data of three different materials (pigskin, pig brain, and human calcaneal fat). The soft tissues were considered to be nonlinear and viscoelastic; the friction coefficient at the contact interface between the specimens and platens was assumed to vary from 0.0 to 0.5. Our numerical simulations show that the tissue specimens are, due to the specimen/platen friction, not compressed in a uniform stress/strain state, as has been traditionally assumed in analytical analysis. The stress of the specimens obtained with the specimen/platen friction can be greater than those with the frictionless specimen/platen contact by more than 50%, even in well-controlled test conditions.     

3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.     

A nanoparticle-based immobilization assay for prion-kinetics study

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint™, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint™ replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.     

Induction of micronuclei in human fibroblasts and keratinocytes by 25 kV x-rays

A relative biological effectiveness (RBE) not much larger than unity is usually assumed for soft x-rays (up to approximately 50 keV) that are applied in diagnostic radiology such as mammography, in conventional radiotherapy and in novel radiotherapy approaches such as x-ray phototherapy. On the other hand, there have been recent claims of an RBE of more than 3 for mammography and respective conventional x-rays. Detailed data on the RBE of soft x-rays, however, are scarce. The aim of the present study was to determine the effect of low-energy x-rays on chromosomal damage in vitro, in terms of micronucleus induction. Experiments were performed with 25 kV x-rays and a 200 kV x-ray reference source. The studies were carried out on primary human epidermal keratinocytes (HEKn), human fibroblasts (HFIB) and NIH/3T3 mouse fibroblasts. Micronucleus (MN) induction was assayed after in vitro irradiation with doses ranging from 1 to 5.2 Gy. Compared to the effect of 200 kV x-rays, 25 kV x-rays resulted in moderately increased chromosomal damage in all cell lines studied. This increase was observed for the percentage of binucleated (BN) cells with micronuclei as well as for the number of micronuclei per BN cell. Moreover, the increased number of micronuclei per micronucleated BN cell in human keratinocytes and 3T3 mouse fibroblasts suggests that soft x-rays induce a different quality of damage. For all cell lines studied the analysis of micronucleus induction by 25 kV soft x-rays compared to 200 kV x-rays resulted in an RBE value of about 1.3. This indicates a somewhat enhanced potential of soft x-rays for induction of genetic effects.     

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.     

Image blurring by thermal diffusion in the observation of hydrated biomolecules with soft X-ray microscopy.

A simple model is presented for the estimation of image blurring in X-ray microscopy of biological specimens in a hydrated environment. The model is essentially based on thermal diffusion of an object to be imaged. The degree of image blurring by diffusion depends on the following situations of the object. The object is free from, is tightly fixed to, or is partially connected to the surrounding structures. The proper imaging time required to achieve a given resolution in X-ray microscopy of biological structures was estimated with the present method. The results suggest that imaging time shorter than 3 msec (free) to 1.4 sec (tightly fixed) is required for the observation of a cell (30 microns in diameter) at the resolution of 100 nm. The model is also applicable to a fragmented object caused by imaging X-rays.     

Nuclear changes in cytologic specimens obtained by CT-guided fine needle aspiration biopsies.

A light and electron microscopic study was conducted to investigate nuclear changes observed in specimens obtained from various sites by computed tomographic (CT)-guided fine needle aspiration (FNA) biopsy. These changes, which consisted of a disappearance of the nuclear chromatin and a disruption of the nuclear membrane, were found in 70% of 10 FNA specimens obtained with CT guidance, in 26% of 15 specimens obtained with fluoroscopic guidance and in 0% of 10 specimens obtained without x-ray imaging techniques. Although the number of cases studied was small and the mechanisms responsible for these changes are not clear, cytopathologists should be aware of alterations in evaluating FNA specimens obtained with the guidance of x-ray imaging techniques.     

Utilization of hydrogen,acetate, and “noncompetitive”; substrates by methanogenic bacteria in marine sediments

We have used a relatively new microscopical technique, environmental scanning electron microscopy (ESEM), along with transmission electron microscopy (TEM) and light microscopy, to investigate a unique microbial community from a temperate-climate, cold sulfide spring near Ancaster, Ontario, Canada. ESEM allows the viewing of fully hydrated specimens that have not undergone the structural or chemical alterations imposed by the extensive procedures necessary for viewing biological specimens in a vacuum. Besides allowing visualization of microorganisms in their natural form and as intact assemblages, ESEM also detects elements, especially those lighter than Si, which tend to be lost or masked by the processes used to prepare samples for conventional SEM and for TEM thin sections. In this study we report new information about the structure of bacteriogenic sulfur deposits and their relationship to the structural aspects of a natural microbial community from a cold sulfide spring.     

Validity and cost-effectiveness of the Gonozyme test in the diagnosis of gonorrhea.

Although bacterial culture is considered to provide the most definitive diagnosis of gonorrhea, it has limitations when specimens must be transported long distances. A study was carried out to evaluate the validity and cost-effectiveness of an alternative method of diagnosing gonorrhea, the Gonozyme test, a commercially available enzyme immunoassay. Urogenital specimens from 100 men and 100 women with symptoms suggestive of or a history of exposure to gonorrhea were tested for the presence of Neisseria gonorrhoeae by means of bacterial culture and for gonococcal antigen with the Gonozyme test. The specimens from the men were also examined by means of microscopy of Gram-stained smears. The sensitivity and specificity of the Gonozyme test with reference to culture results were 95.6% and 97.4% respectively in the men and 84.2% and 98.7% in the women. The predictive value of a positive result was 91.6% in the men and 94.1% in the women, and the predictive value of a negative result 98.6% in the men and 96.3% in the women. The cost-effectiveness of the Gonozyme test was higher than that of bacterial culture in this population, which had a high prevalence rate of gonorrhea (23% in the men and 19% in the women). The Gonozyme test would be an adequate alternative to culture for the diagnosis of gonorrhea and contact tracing in areas far from diagnostic laboratories.     

Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces.

Adhesion and cytoskeletal structure are intimately related in biological cell function. Even with the vast amount of biological and biochemical data that exist, little is known at the molecular level about physical mechanisms involved in attachments between cells or about consequences of adhesion on the material structure. To expose physical actions at soft biological interfaces, we have combined an ultrasensitive transducer and reflection interference microscopy to image submicroscopic displacements of probe contact with a test surface under minuscule forces. The transducer is a cell-size membrane capsule pressurized by micropipette suction where displacement normal to the membrane under tension is proportional to the applied force. Pressure control of the tension tunes the sensitivity in operation over four orders of magnitude through a range of force from 0.01 pN up to the strength of covalent bonds (approximately 1000 pN)! As the surface probe, a microscopic bead is biochemically glued to the transducer with a densely-bound ligand that is indifferent to the test surface. Movements of the probe under applied force are resolved down to an accuracy of approximately 5 nm from the interference fringe pattern created by light reflected from the bead. With this arrangement, we show that local mechanical compliance of a cell surface can be measured at a displacement resolution set by structural fluctuations. When desired, a second ligand is bound sparsely to the probe for focal adhesion to specific receptors in the test surface. We demonstrate that monitoring fluctuations in probe position at low transducer stiffness enhances detection of molecular adhesion and activation of cytoskeletal structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of design variables of condylar total knees on the joint forces in step climbing based on a computer model.

The ability to climb a steep step or rise from a low chair after total knee replacement may be enhanced if the required force in the quadriceps muscle is reduced. This can potentially be achieved if the total knee produces a large lever arm measured from the femoral-tibial contact point to the patellar ligament. A reduced quadriceps force would also reduce the patello-femoral force and the femoral-tibial contact force. The contact point location is likely to be a function of the geometry of the femoral and tibial components in the sagittal plane, including the relative distal and posterior radii of the femoral profile, the location of the bottom-of-the-dish of the tibial surface, the radius of the tibial surface, and the presence or absence of the posterior cruciate ligament. A three-dimensional model of the knee was developed including the quadriceps and various ligaments. In the study, the motion was confined to flexion extension and displacement in the sagittal plane. The quadriceps was assumed to be the only muscle acting. A standard software package (Pro/Mechanica) was used for the analysis. For a femoral component with a smaller distal radius, there was 12% reduction in the quadriceps muscle force and up to 11% reduction in the patello-femoral force from about 100 up to 60 degrees flexion. However, apart from that, there were less than 10% differences in all the forces as a function of all of the design variables studied. This was attributed to the relatively small changes in the lever arm of the patella tendon, since the tendon moves in an anterior-posterior direction along with the femur. An additional factor explaining the results was the change in the anterior-posterior contact point as controlled by the forces in the patella tendon and in the soft tissues. The results imply that for a standard condylar replacement knee, the muscle and contact forces are not greatly affected by the geometrical design variables.