Showing papers in "Journal of Microscopy in 1996"

New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy

Abstract: Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts. With the atomic force microscope we are able to study the structure of the asphalts without any pre-preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method. In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.

Two-photon excited lifetime imaging of autofluorescence in cells during uva and nir photostress

Abstract: By monitoring coenzyme autofluorescence modifications, as an indicator of cell damage, the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UVA radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser, focused through high-numerical-aperture microscope optics, provided diffraction-limited microbeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (tau-mapping). Upon the onset of UV or NIR exposure, Chinese hamster ovary cells exhibited blue/green autofluorescence with a mean lifetime of 2.2 ns, which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 mW, 300 J cm-2) resulted in oxidative stress correlated with increased autofluorescence intensity, onset of nuclear fluorescence, and a fluorescence lifetime decrease. The cellular response to femtosecond NIR microbeams depended significantly on peak power. Peak powers above a threshold value of about 0.5 kW (average power: 6 mW). 0.55 kW (7 mW) and 0.8 kW (10 mW) at 730 nm, 760 nm and 800 nm, respectively, resulted in the onset of short-lived luminescence with higher intensity (100 x) than the intracellular NAD(P)H fluorescence. This luminescence, accompanied by destruction of cellular morphology, was localized and occurred in the mitochondrial region. In contrast, beams at a power of less than 0.5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.

Brefeldin A effects in plant and fungal cells: something new about vesicle trafficking?

Abstract: Whilst the function and organization of the secretory machinery in eukaryotic cells exhibit basic similarities, the compartmentation of the endomembrane system can show significant differences between the fungal, plant and animal kingdoms. The use of the antibiotic brefeldin A (BFA) as an inhibitor of secretion in both animal and yeast cells has resulted in a remarkable advance in our understanding of the modes of action of vesicle shuttles between the endoplasmic reticulum and Golgi apparatus and within the Golgi apparatus itself. It is now apparent that application of the drug to filamentous fungi and plants will also help unravel the workings of the secretory system in these organisms. In this paper we review recent progress in our laboratories on elucidating the effects of BFA on the morphology of the Golgi apparatus and compare these with recently published data on fungal and plant cells. Variation in the response to BFA are reported, which may not all be attributed to differences in drug concentration and time of treatment. These may reflect differences in cellular sensitivity or multiple sites of action of the drug, and the existence of a specific molecular target for BFA is questioned.

‘Pod shatter’ in Arabidopsis thalianaBrassica napus and B. juncea

Abstract: Wild Brassica plants release seeds by a pod shattering mechanism; in related crop plants, such as oil-seed rape, this can result in substantial loss of seed, and hence loss of revenue, and also in the distribution of seeds which can contaminate future crops and the environment. To identify strategies which may be used to reduce shatter, either by conventional breeding programmes or by genetic engineering, we have examined fruit development in oil-seed rape (Brassica napus), and in the related B. juncea and Arabidopsis, using a combination of cytological, cytochemical and molecular techniques. We report here on the patterns of cellular differentiation and tissue development during fruit maturation, and suggest how this results in the shattering phenotype.

Determination of the size distribution of lecithin liposomes: a comparative study using freeze fracture, cryoelectron microscopy and dynamic light scattering

Abstract: The size distribution of liposomes is often determined using freeze fracture, cryoelectron microscopy or dynamic light scattering. However, the resulting size distributions do not directly coincide owing to the different weighting of the techniques. We present several methods which correct for these effects and allow a comparison of liposome size distributions as obtained by freeze fracture, cryoelectron microscopy or dynamic light scattering. These methods are based on theoretical models for the weighting of the size distribution of liposomes, which result from the preparation procedure for freeze fracture electron microscopy and from the measurement by dynamic light scattering. The proposed transformation methods are then experimentally tested with a sample of lecithin liposomes, whose size distribution was determined by dynamic light scattering, freeze fracture and cryoelectron microscopy. Furthermore, the weaknesses of the experimental techniques and hence of the resulting size distributions are discussed.

Fourier transform multipixel spectroscopy for quantitative cytology

Abstract: A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104 pixels in a single cell. The net result was I(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food-digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.

Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system

Abstract: The bilateral imaging approach known from confocal applications operating in the line mode was used to realize real-time two-photon imaging. It is shown that the sectioning inherent to two-photon imaging could be improved by the introduction of a confocal line aperture in the imaging path. Using a high-power, low-repetition-rate amplified Ti:sapphire system, various biological objects were visualized including live boar sperm.

Aberration control in quantitative imaging of botanical specimens by multidimensional fluorescence microscopy

Abstract: Three dimensional (3-D) fluorescence microscopes, including conventional instruments with digital deblurring, confocal systems and two-photon excitation, all exhibit monochromatic and chromatic aberrations. A simple Gaussian model of the aberrated point spread function and optical field for each instrument illustrates spatial distortions and blurring and some unique attenuation effects in confocal and two-photon microscopy. These properties depend on the manner in which illumination and detection combine to give the overall microscope performance and highlight the importance of both optics and sample aberrations; the specimen must be considered as an optical component of the integrated imaging system. Axial focus distortion, from refractive index boundaries at the sample, can be accurately modelled by a geometric ray tracing programme, considering weighted components across the entire objective lens working NA. The modal z-focus error, rather than the average of all weighted rays, agrees closely with empirical measurements of axial focus position from test samples. This agreement is particularly close for confocal measurements when NA4 weighting is used in the model calculations, but the situation is more complex for samples with non-planar refractive boundaries. Calibration of axial attenuation in a botanical sample, arising from the combination of optical sectioning with specimen-induced spatial distortions and blurring, is possible using an in situ fluorescence sea within a permeabilized preparation. Parametric descriptions of attenuation can be obtained through the guard cell complex of Commelina communis leaf epidermis. Improved images of the 3-D morphology of stomatal guard cells are then obtained by digital correction of attenuation and spatial distortion. Calibrations can be routinely used to correct experimental data by integration with a structured image file format.

Dispersion, aberration and deconvolution in multi-wavelength fluorescence images

Abstract: The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi-wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.

Morphological studies of pseudowollastonite for biomedical application.

Abstract: Pseudowollastonite ceramic (psW) composed of CaO.SiO2 was found to be bioactive in a simulated body fluid environment. The chemical reaction initiated at the material surface resulted in hydroxyapatite (HA) formation. These bone-bonding properties are essential for securing the necessary physico-chemical integration of the material with living bone. Materials behaving in this way can be considered for potential biomedical application as bone tissue substitute for a natural bone repair or replacement as implant. A mechanism of hydroxyapatite formation on pseudowollastonite ceramics surface was investigated during exposure to a stimulated body fluid (SBF) for a period of 3 weeks. Morphology and structure of the surface product and its original substrate was examined by thin-film X-ray diffraction, scanning electron microscopy and high-resolution transmission electron microscopy. HA crystals were found to form on an amorphous silica intermediate layer. (100) lattice planes of HA were resolved and identified. Concentration of ions in the SBF and pH of the SBF were monitored throughout the exposure. Additional pH measurements were made at the interface of psW with SBF. The HA formation occurred when there was a sudden increase of pH from 7.25 to 10.5 at the interface of psW with SBF as a result of ionic exchange between 2H+ and Ca2+ within the psW network. This ionic exchange transformed the psW crystals into an amorphous silica phase. The appropriate pH and the ion concentrations were essential for partial dissolution of the amorphous silica phase and subsequent precipitation of a Ca-P rich phase which then transformed to HA.

Membrane deformation of living glial cells using atomic force microscopy

Abstract: Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epifluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force-distance curves indicated that standard tips did not penetrate the membrane while sharper Supertips TM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.

High-pressure freezing of tissue obtained by fine-needle biopsy.

Abstract: High-pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze-substitution) of biological tissue up to a thickness of about 200 microns. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine-needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.

4Pi-confocal images with axial superresolution

Abstract: Summary We present two-photon excitation 4Pi-confocal images of clustered fluorescence beads demonstrating threedimensional far-field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four-fold improved axial resolution is achieved by engineering the point-spread function through a suitable combination of aperture enlargement, two-photon excitation, confocalization and three-point deconvolution. In contrast to their confocal counterparts, 4Pi-confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi-confocal images corresponds to about one-fifth of the wavelength and surpasses the lateral resolution by 25%.

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy

Abstract: Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells. Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 microns from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.

The relative precision of crystal orientations measured from electron backscattering patterns

Abstract: Recently developed statistical methods for analysing orientation data are presented and applied here in a study of the precision by which crystal orientations can be measured from electron backscattering patterns. The use of these methods allows a direct comparison to be made between the precision obtained with manually and automatically localized bands, which is important owing to a more and more widespread use of fully automatic analysis of electron backscattering patterns. Curves which show how the precision depends on the pattern quality and on the number of bands used for the orientation measurements are presented for both manually and automatically localized bands. Typical values for the relative precision of crystal orientations measured from electron backscattering patterns are shown to be of the order of 0.5° for manually localized bands and 0.75° for automatically localized bands, when about 10 bands are used for the measurements. In a more realistic situation where a careful operator is willing to localize four to five bands in each pattern, the precision of the measured crystal orientations is similar to that obtained for automatically localized bands.

Methanol as a rapid fixative for the investigation of plant surfaces by SEM

Abstract: A rapid fixation/dehydration method of plant specimens for scanning electron microscopy is presented. Prior to critical-point drying (CPD) the specimens are immersed in pure methanol. Methanol incubation instantly fixes the elastically extended cell walls. Owing to this instant fixation, shrinking of the specimens is prevented, resulting in an improved preservation of cell dimensions comparable to in vivo conditions. The method is most suitable for plant epidermal surfaces. It avoids the time-consuming fixation/dehydration in routine investigation of plant surfaces prior to CPD, especially for delicate specimens.

Multivariate statistical analysis of feg‐stem edx spectra

Abstract: A multivariate statistical analysis method has been used to examine the information content of a series of EDX spectra acquired using a FEG-STEM recorded across a segregated grain boundary in a ferritic steel. The spectra have been factored into a set of orthogonal information components (eigenspectra), each of which corresponds to an independent source of information. Two meaningful eigenspectra are identified: one is related to the segregation of P and Cr and the other to a combination of self-absorption and coherent bremsstrahlung. Spectra are reconstructed from the meaningful eigenspectra to reduce noise and improve the sensitivity for detection of trace element segregation.

Analysis of efficiency of two-photon versus single-photon absorption for fluorescence generation in biological objects

Abstract: Starting from basic absorption cross-section values and breakdown limitations on possible field strengths it is shown that the total exposure required for equivalent fluorescence generation by single-photon absorption (SPA) is at least an order of magnitude lower than by two-photon absorption (TPA). The difference is such that TPA may in fact in many cases offer no advantage over SPA with respect to the damage induced during fluorescence imaging of biological materials.

A simple cryo‐SEM method for delicate plant tissues

Abstract: A simple modification to the existing preparative procedures for examining delicate plant tissues and insect–plant interactions by cryo-SEM is described. Samples are attached to a stub with colloidal graphite, introduced into the air lock, evacuated briefly, then transferred onto the cryo-stage of the preparation chamber where full evacuation and slow cooling take place. After sputter coating with gold, the samples are transferred onto the microscope cold stage and examined at 15 kV.

Gibberellic-acid-induced reorientation of cortical microtubules in living plant cells

Abstract: By microinjecting rhodamine-conjugated porcine tubulin into pea epidermis we recently showed how cortical microtubules reorientate from transverse to longitudinal in living cells (Yuan et al., 1994, Proc. Nat. Acad. Sci, USA91, 6050–6053). In the present paper we compare this reorientation with the contrary longitudinal to transverse realignment induced by adding gibberellic acid to pre-injected cells on the microscope slide. Both kinds of reorientation are initiated by the appearance of ‘discordant’ microtubules which do not share the existing alignment but anticipate the new direction. These increase in number as the existing microtubules depolymerize, one alignment apparently replacing the other in a continuous process. By rotating stacks of confocal sections by computer methods we have previously shown that microtubules at the outer tangential cell wall do not necessarily have the same orientation as microtubules at the adjoining anticlinal walls of the same cell (Yuan et al., 1995, Plant J.7, 17–23). This suggests that microtubule reorientations in these epidermal cells occur mainly (or, at least, first) at the outer wall, indicating that the array may not reorientate as a whole. Collectively, these data emphasize the discontinuous nature of the realignment process, the importance of new microtubule polymerization, and the special property of the outer epidermal surface as a sensitive domain.

Cortical microtubule reorientation in higher plants: dynamics and regulation

Abstract: This paper aims to review aspects of cortical microtubule reorientation in higher plant cells. First, we look at the divergent environmental and developmental signals that can elicit the realignment of microtubules in interphase cells. Second, the regulatory factors that might orchestrate microtubule reorientation are examined. In particular, we address the questions of how these extracellular signals are perceived, by what mechanisms this information might be transmitted to the cortical microtubules, and what molecular factors regulate the process of realignment. We put forward an hypothesis of how electric fields reorientate microtubules in plant cells, focusing on the role of transmembrane proteins which might link cortical microtubules in the cytoplasm to the extracellular matrix. Finally, the need to examine microtubule reorientation in live cells is discussed, and we describe the novel visualization of microtubules in live cells of an intact plant. We conclude with our perspective of the future path of research which will be necessary to broaden our understanding of how microtubules undergo rapid reorientation in plant cells.

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Abstract: Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference. AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

Study of 'wet' polymer latex systems in environmental scanning electron microscopy: Some imaging considerations

Abstract: Environmental scanning electron microscopy has been used to study 'wet' polymer latex films. Comparison of the results obtained by imaging in nitrogen and nitrous oxide gas and water vapour has provided insight into a variety of different contrast phenomena. Notably, it was found that edge definition and fine features could be enhanced by imposing further saturated vapour conditions at the hydrated film surface. Furthermore, by imaging at subzero temperatures in the presence of nitrogen, it was shown that beam damage could be reduced in such sensitive polymeric samples.

Zero-loss energy filtering under low-dose conditions using a post-column energy filter

Abstract: Electron cryomicroscopy combined with energy filtering can be performed under low-dose conditions using a post-column energy filter. The microscope combined with the filter is set up such that it can be used with similar ease as a conventional microscope, the main difference being that all filter and microscope control is performed through a central computer and images are recorded with a cooled slow-scan CCD camera. The microscope can also still be used for regular imaging on film as without the filter. Owing to the 18 times post-magnification of the filter, the microscope normally has to be operated at a small magnification, e.g. 3000×, and the beam has to be contracted to a small spot, e.g. 5 mm, in the plane of the microscope viewing screen. Computer control allows one to perform a variety of tasks automatically, such as autofocusing, thickness measurements, most-probable-loss imaging, CCD spot-scanning and tomography. The gain in contrast due to zero-loss energy filtering is analysed using visual inspection, power spectra and Fourier ring correlation. The thickness range for ice-embedded specimens in which a filter at 120 kV is most useful appears to be between 100 and 300 nm.

Ultrastructure and composition of the cell surfaces of infection structures formed by the fungal plant pathogen Colletotrichum lindemuthianum

Abstract: A variety of microscopical techniques and molecular probes have been used to study the ultrastructure and composition of the cell surfaces of the conidia (i.e. spores) and infection structures produced by the hemibiotrophic fungal plant pathogen Colletotrichum lindemuthianum. The fungal conidium germinates to produce a germ-tube, the tip of which swells to produce a domed, melanized appressorium which adheres firmly to the plant surface. Penetration of the cuticle and cell wall is followed by the development of a biotrophic intracellular hypha, which is surrounded by an invagination of the host plasma membrane. Freeze-substitution of C. lindemuthianum germlings showed that conidia are coated with a dense layer of fibrillar material. This ‘spore coat’ contains irregularly shaped pores, giving it a reticular appearance. Negative staining of germlings revealed the presence of numerous long, flexuous fibres or fimbriae, protruding from the surfaces of germ-tubes and appressoria. Colloidal gold was used to visualize fungal extracellular proteins. The colloidal gold stained a fibrillar sheath around germ-tubes, whereas appressoria were surrounded by a halo, comprising an inner unstained region and a stained perimeter. The carbohydrate composition of the cell surfaces of the conidia and infection structures was studied by labelling cells with rhodamine- and fluorescein-conjugated lectins. The results showed that the extracellular matrices of germ-tubes and appressoria are very similar in composition, but differ from those of conidia and intracellular hyphae. Monoclonal antibodies have been prepared to germlings and infection structures of C. lindemuthianum and their use has provided further evidence that the extracellular matrices around germ-tubes and appressoria have several glycoproteins in common. The results also show that the cell surface of C. lindemuthianum becomes specialized during biotrophic development inside host cells.

Fluorescence in situ hybridization on human metaphase chromosomes detected by near‐field scanning optical microscopy

Abstract: Fluorescence in situ hybridization o­n human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments o­n a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Comparison of slam-freezing and high-pressure freezing effects on the dna cholesteric liquid crystalline structure

Abstract: Using in parallel electron microscopy of ultrathin frozen-hydrated sections and freeze-fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam-freezing at liquid helium temperature and high-pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam-freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high-pressure freezing does not preserve the structure of the liquid crystal: the long-range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high-pressure freezing process.

Stereological analysis of the layered collagen of human intracranial aneurysms.

Abstract: Intracranial saccular aneurysms are balloon-like distensions of the arterial wall; they increase in size gradually, a few to the point of bleeding or catastrophic rupture. Collagen is the primary structural component of the aneurysmal wall, and because only a small fraction of aneurysms fail, the collagen fabric must effectively reorganize in order to maintain mechanical integrity as an aneurysm changes size. Data were obtained from four human aneurysms, fixed at 110 mmHG of distending pressure with 10% buffered formalin, and sectioned completely through at 4 microns thickness. Each set of measurements included groups of data taken layer by layer from a radial corridor across the aneurysm wall. Each three-dimensional orientation measurement, for which we used a Zeiss polarizing microscope with a universal stage attachment, is defined by an azimuth and elevation angle relative to the section plane. We compared the interdependence of these measured angles with a mathematical model based on fibres following great circle trajectories, and related the measured azimuth and elevation angles to the relative depth of the section into the aneurysm. Data were plotted on Lambert equal-area projections, along with the theoretical relation between azimuth and elevation, that included wall thickness and depth of sectioning. The graphical relationship between measured azimuth and elevation for collagen fibres across the layered fabric of the aneurysmal wall is consistent with the theoretical great circle trajectories for collagen fibre alignment. Analysis was based on statistics for spherical data to give values for the mean orientation and the circular standard deviations (CSD) about that mean. The results indicate that any given region on the aneurysm wall is made up of many, very thin sublayers, most of which have a relatively coherent organization (mean CSD 8 degrees). These measurements agree well with the mathematical model and, when considered collectively, the layers provide a balanced distribution for bearing the biaxial tensile stress of the wall.

Mapping of ELNES on a nanometre scale by electron spectroscopic imaging

Abstract: The amorphous interfacial layer between Si substrates and diamond films grown by plasma-assisted chemical vapour deposition has been studied by electron spectroscopic imaging. The amorphous layer consists mainly of carbon, which can only be distinguished from the diamond film by analysis of the near-edge structure (ELNES) of the carbon K edge. Series of electron spectroscopic images were acquired across the carbon K edge and were analysed in order to reveal the presence of the π*- and σ*-excitations. After background removal from the corresponding images, phase maps for the distribution of sp2 and sp3 hybridized carbon can be obtained. From the whole series of images, electron energy-loss spectra can be extracted for any given area in the images. The results show that the amorphous layer covers large areas along the interface and that regions with only 1–2 nm layer thickness can clearly be analysed. The results obtained with the electron spectroscopic imaging technique will be compared with results obtained on a field emission gun scanning transmission electron microscope.