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At least 109 records · Page 6Linked to original sources
PubMed1979

Scanning electron microscopy at macromolecular resolution in low energy mode on biological specimens coated with ultra thin metal films.

In this report, conditions for attaining high resolution in scanning electron microscopy with soft biological specimens are described using the currently available high resolution scanning electron microscopes in emission mode of low energy electrons (secondary and charging electrons). Retinal rod outer segments, red blood cells, intestinal mucosa, and ferritin molecules were all used as biological test specimens. From uncoated specimens a new source of signal, referred to as a discharge signal, can provide a high yield of low energy electrons from an excitation area approximately the size of the beam's cross section. Additionally, under these conditions sufficient topographic contrast can be achieved by applying ultra thin metal coatins. A 0.5 nm thick gold film is found sufficient for generating the total signal, whereas increased coating thickness causes additional topographic background signal. However, a 2.0 nm film is needed for imaging surface details with the present instrument. Ultra thin, even, and grainless tantalum films have been found effective in eliminating the charging artifacts caused by external fields, and the decoration artifacts caused by crystal growth as seen in gold films. To improve, in high magnification work on ultra thin coated specimen, signal-to-noise ratio, methods for obtaining saturation of the signal with discharge electrons are shown. The necessity of confirming the information obtained in SEM by independent techniques (TEM of stereo-replicas or ultra thin sections) is discussed.

Animals↗
PubMed1997

High resolution and high fields in biological solid state NMR.

2H solid state NMR spectra of a polypeptide in an oriented membrane environment is demonstrated to have an orientational resolution of 0.3 degree. Such data results in high resolution structural constraints. Similar spectra are demonstrated at 23.2 T using a resistive magnet at the National High Magnetic Field Laboratory.

Anti-Bacterial Agents↗
PubMed2003

Modern laser scanning microscopy in biology, biotechnology and medicine.

Laser microscopic techniques currently used in morphology and cell biology represent highly sensitive tools for detecting biomolecules within their natural environment. Use of the fluorescence-, reflectance- and transmission modes of confocal laser scanning microscopes (CLSM) equipped with He-Ne- and Ar+-ion lasers for CeIV and DAB based detection of endogenous or immunobound enzymatic activities in tissue sections (vibratome, cryostat, paraffin and semithin plastic sections) opens a wide range of interesting new possibilities in cellular and molecular biology. Increased resolution power, blur-free confocal imaging, higher sensitivity, optical sectioning capability and 3D-image analysis provide a large quantity of valuable information about biological objects specimens. The new infrared multiphoton laser scanning microscopy (NIR-LSM) is increasingly becoming the optical tool of choice for (a) fluorescence imaging of cellular and subcellular components with high spatial and temporal resolution, (b) fluorescence resonance energy transfer between physiologically relevant molecular species involving protein-protein interactions, (c) nanoprocessing within living cells and tissues, with varied applications in (d) photochemistry and (e) medical diagnostics as well. Both, CLSM and NIR-LSM as modern microscopical strategies are indispensable in basic research and will prove to be invaluable for clinical diagnostic studies and therapy in the near future.

Animals↗
PubMed1991

Conventional and high resolution scanning electron microscopy of biological sectioned material.

Intracellular structures of embedded biological tissues (rat kidney, myocardium and small intestine) were observed by conventional-scanning electron microscopy (C-SEM) and high-resolution scanning electron microscopy (HR-SEM) after glass knife sectioning. C-SEM of semi-thin sections of material processed the same way as conventional transmission electron microscopy (TEM) provided strong backscattered electron (BSE)-dependent, two-dimensional secondary electron images (SEI(-)) which precisely integrated and further extended previous light microscopy (LM) observation of the same specimen. In addition, the three-dimensional (3-D) arrangement of intracellular organelles was appreciated using a mixture of acetone-soluble acrylic resin in place of epoxy resin embedding. Since the identification of such structures was hampered by the use of conventional fixations we introduced osmium maceration as a preliminary step to remove excess cytoplasmic matrix from the specimen. Consequently, semi-thin sections for LM and thin sections for TEM were obtained by sectioning of the tissue blocks. After resin removal, the sections were successfully observed in 3-D under a C-SEM. Finally, the deembedded, osmium treated sections proved to be smooth enough to facilitate deposition of continuous, ultra-thin (1 nm) chromium films and, therefore, HR-SEM studies of macromolecular cell membrane structures.

Animals↗
PubMed1997

An account of NMR in structural biology.

With the ability to determine atomic resolution structures of biological macromolecules in semi-physiological conditions, nuclear magnetic resonance spectroscopy (NMR) has become an eminent tool in structural biology. NMR provides a means for studying critical biological phenomena including protein structure, dynamics and folding as well as a practical approach to drug design.

Crystallography, X-Ray↗
PubMed1997

High resolution electron paramagnetic resonance imaging of biological samples with a single line paramagnetic label.

The application of electron paramagnetic resonance imaging (EPRI) to obtain information from biological samples has been limited by the lack of ideal single line radical labels. The commonly used nitroxides exhibit multiple lines causing either hyperfine-based limitations in the maximum obtainable image resolution or hyperfine-based artifacts in the reconstructed image. The use of a novel single-line triarylmethyl paramagnetic label that enables marked enhancement in image quality and resolution is reported. This label exhibits a single line EPR spectrum that is sharp (linewidth approximately 60 mG in the absence of oxygen) and relatively stable in tissues. The potential of this label in enabling high resolution EPR imaging of biological samples was demonstrated in a series of phantoms and isolated biological organs such as the rat kidney. The images demonstrate that resolutions better than 100 microns could be obtained at L-band on samples of up to 20 mm in size.

Animals↗
PubMed2002

Towards high-resolution 1H-NMR in biological membranes: magic angle spinning of bicelles.

Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment.

Animals↗
PubMed1975

Microscopic tracing of deuterium.

The development of the ion microprobe analyser has made it possible to follow stable isotopes with microscopic resolution in biological and other materials. An ion microprobe using an oxygen primary beam has been found to produce relatively few mass 2 ions (+ or -) on organic samples. This technique should permit the use of deuterium as a practical tracer with a resolution of about 1 mum in the plane of the section and 2 nm in the depth dimension with available instruments.

Animals↗
PubMed1995

A biological basis for instantaneous centres of rotation of the vertebral column.

The instantaneous centre of rotation has proven to be a useful parameter of vertebral motion. The normal location of instantaneous centres has been determined in cadavers and in normal volunteers for the cervical, thoracic and lumbar spines, and abnormal location of centres has been shown to correlate with spinal pain. However, to date, an instantaneous centre has constituted no more than a convenient mathematical summary of vertebral kinematics. It has defied resolution into biologically meaningful parameters. This study offers a novel model of vertebral motion in which the instantaneous centre of rotation can be shown to be a function of the location of the centre of reaction of a vertebra, and the intrinsic rotation and translation it undergoes. These parameters are strictly linked by equations that determine the location of an axis of rotation. These equations allow aberrations in the location of an axis to be interpreted in terms of the anatomical and pathological factors that affect the centre of reaction of the vertebra and the rotation and translation it undergoes.

Biomechanical Phenomena↗
PubMed1987

The use of a charge-coupled device for quantitative optical microscopy of biological structures.

The properties of a charge-coupled device (CCD) and its application to the high-resolution analysis of biological structures by optical microscopy are described. The CCD, with its high resolution, high sensitivity, wide dynamic range, photometric accuracy, and geometric stability, can provide data of such high quality that quantitative analysis on two- and three-dimensional microscopic images is possible. For example, the three-dimensional imaging properties of an epifluorescence microscope have been quantitatively determined with the CCD. This description of the imaging properties of the microscope, and the high-quality image data provided by the CCD, allow sophisticated computational image processing methods to be used that greatly improve the effective resolution obtainable for biological structures. Image processing techniques revealed fine substructures in Drosophila embryonic diploid chromosomes in two and three dimensions. The same approach can be extended to structures as small as yeast chromosomes or to other problems in structural cell biology.

Animals↗
PubMed1989

The case for low voltage high resolution scanning electron microscopy of biological samples.

Dried biological samples are low in scattering power, non-conducting and sensitive to radiation damage. These facts complicate the choice of the optimum beam voltage Vo at which they should be observed in the scanning electron microscope (SEM) because they add as variables the type and thickness of the coating material and degradation/contamination of the specimen by the beam. Heretofore, high resolution SEM could only be carried out at relatively high Vo (20-30kV) because available equipment could not produce small beam diameters at low Vo. Modern instruments can produce beam diameters of about 3nm at 1.5kV. As normal preparative procedures (fixation, critical point drying, coating) are unlikely to preserve reliable structure below this level, it is now possible to investigate the possible advantages associated with low Vo operation such as a reduction in charging and radiation damage and improved topographic contrast. The conclusion recommended by this paper is that the term resolution needs careful definition. The size of the smallest features visible in a micrograph is a function of many variables. Although probably the most important is specimen preparation, a number of others (probe size, beam penetration range, contamination, coating thickness needed to provide contrast and avoid charging etc) are functions of Vo. Of these variables at least probe size and possibly contamination become more favorable at higher Vo while the remainder favor low Vo. As a result the optimum will occur at a Vo where the best balance of these factors occurs for a particular sample. When using the Hitachi S-900, we have found that the optimum seems to be at 1.5-2.5kV for topologically diverse samples, but may extend to 5kV on samples on which very small structural details have been preserved and which are relatively stable to radiation damage.

Animals↗
PubMed1997

Videodensitometric analysis of electron spectroscopic micrographs--a tool for detection of biologically relevant elements with high resolution.

Electron energy-loss spectroscopic imaging (ESI) yields high-resolution, element-sensitive images. However, ESI suffers from difficulties in distinguishing element-specific and background contributions. New methods have therefore been introduced which use grey-level measurements in micrographic images for a more accurate detection of element distributions. A videodensitometric method allowed the detection of low phosphorus levels in axoplasmic neurofilaments of squid giant axons. Here we further verify these results by investigating the relationship of videodensitometry and electron energy-loss spectroscopy (EELS), particularly considering the peculiarities of these methods in terms of automatic background correction and representation of the results. Six biological specimens and two nonbiological specimens were examined both by EELS and by videodensitometry. In all cases comparable results were obtained. The overlapping PL2,3 and SL2,3 ionization edges could clearly be recognized individually by both methods, and controls showed that mass density variations within the specimens did not impair elemental analysis. Additional evidence supporting the detection of phosphorylation sites in squid neurofilaments was obtained in both EELS and videodensitometric measurements of neurofilament-enriched pellets and of aggregated axoplasmic particles. Thus, video-densitometry appears to be a useful tool for an improved exploration of the full imaging capabilities of energy filtering electron microscopy.

Animals↗
PubMed1997

The first glimpse of a complex of nitrogenase component proteins by solution X-ray scattering: conformation of the electron transfer transition state complex of Klebsiella pneumoniae nitrogenase.

An essential feature of the mechanism of nitrogenase, the enzyme responsible for biological nitrogen fixation, is the formation of a transient electron transfer complex between the MoFe protein containing the active site at which N2 is reduced, and the Fe protein, which functions as a specific electron donor to the MoFe protein. We have obtained high quality solution X-ray scattering data using synchrotron X-rays of a stable putative electron transfer complex, (MoFe-protein)(Fe-protein.ADP.AIF4)2, of Klebsiella pneumoniae and used the model-independent approach based on the multipole expansion method to provide a stable and unique shape restoration at approximately 15 A resolution. The biological significance of this first molecular structure of a nitrogenase complex is discussed.

Azotobacter vinelandii↗
PubMed1999

Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning.

Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM. Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing. Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face. The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces. The cytoplasms show particles of different size which most likely represent proteins. The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging. High quality cryo-sections are only obtained from vitrified material. Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.

Animals↗
PubMed1982

Stereochemical studies of demethylated ketamine enantiomers.

The enantiomorphs of norketamine, 2-(o-chlorophenyl)-2-aminocyclohexanone, were synthesized and screened for biological activity. Resolution was achieved by fractional crystallization of the tartrate salts. Stereochemical purity was determined using standard GC or GC-MS analysis. Preliminary pharmacological evaluations revealed that intraperitoneally injected dextrorotatory norketamine caused a greater duration of loss of righting reflex in mice than the levorotatory isomer.

Anesthetics↗
PubMed1993

Resolution enhanced NMR spectroscopy in biological systems via magnetic susceptibility matched sample immersion chambers.

A technique is described which reduced the magnetic susceptibility induced line broadening in NMR spectra obtained from three biological systems at 4.7 Tesla. Proton spectra from a sealed suspension of HL60 leukemic myeloblasts yielded minimum linewidths of 1.3 Hz at 200 MHz (0.0065 ppm) after 10 min of automated shimming. 31P spectra from an in vivo murine MCa mammary carcinoma yielded a well-resolved phosphorylcholine resonance without proton decoupling and with the resistive shim coil currents set to zero. 31P spectra from a perfused suspension of RIF-1 fibrosarcoma cells exhibited a gamma-nucleoside triphosphate resonance which was resolved into purine and pyrimidine components.

Animals↗