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Biomedical subjects

R M Glaeser

Publications and source records attributed to R M Glaeser.

At least 55 records · Page 3Linked to original sources
PubMed1981

Periodic surface array in Caulobacter crescentus: fine structure and chemical analysis.

A periodic array structure on the cell surface of Caulobacter crescentus CB15 was revealed by electron microscopy of the cell envelope, using negative staining, thin-sectioning, and freeze-etching. This structural layer has been isolated from liquid cultures, in which large pieces of the two-dimensional array are shed by cells grown to high density. Often areas of intact array corresponding to the entire cell surface could be found. The hexagonally arranged structure was highly ordered and had an unusual degree of complexity, as determined by optical diffraction and computer processing of micrographs of negatively stained, isolated surface array. Filtered, reconstructed images were obtained from both normal and low-electron-dose micrographs demonstrating resolutions of 2.9 and 25 nm, respectively. Comparison by optical diffraction and image filtering of micrographs recorded by using either normal or minimal beam exposure techniques suggested that the lower-resolution features of the image are very stable to electron exposure. Gel electrophoresis indicated that isolated array preparations contain a number of polypeptides. It appears likely that more than one of these proteins are structural components of the array, in contrast to a single protein found in many bacterial surface arrays. The Caulobacter surface array is also unusual in that the repeated units are widely spaced with no apparent direct connection. Computer spatial averaging provided information about the shape and complexity of the connecting elements, and this was compared with some additional electron microscopic evidence of linking structures. Thin-sectioning studies confirmed the image features seen by other techniques, but the addition of tannic acid in the fixation procedure was required to visualize the structure. A comparison of these results with out current knowledge of the Caulobacter cell envelope suggests interesting questions about the biogenesis of this membrane structure and its involvement in the cell development process of this organism.

Bacteria↗
PubMed1980

High resolution cold stage for the JEOL 100B and 100C electron microscopes.

A high resolution, liquid nitrogen-cooled specimen stage has been designed and constructed for use on the JEOL 100B and 100C electron-microscopes. This stage will be useful for imaging biological macromolecular arrays in the frozen-hydrated or glucose-embedded states at low temperature. Images thus obtained should have an increased signal-to-noise ratio due to the radiation damage protection offered by low temperature.

Cell Membrane↗
PubMed1980

Electron diffraction from single crystals of DNA.

Crystals of DNA have been grown in a form suitable for study by electron diffraction and electron microscopy. Preliminary electron diffraction patterns of any type that have been obtained from single crystals of highly polymerized DNA. The patterns, obtained from frozen, hydrated crystals with the beam approximately parallel to the DNA strand axis, show a hexagonal geometrical arrangement with a (1,0) Bragg spacing of 23.1 A.

Animals↗
PubMed1979

Radiation damage of purple membrane at low temperature.

The intensities of diffracted electron beams for the purple membrane of Halobacterium halobium are found to decay exponentially as a function of the accumulated electron exposure, both at room temperature and at -120 degrees C. This permits us to define the "critical dose" Ne(h,k) for the (h,k) diffracted beam, as being the electron exposure (electrons/A2) at which the diffracteed intensity has fallen to e-1 of its initial value. The critical of purple membrane is found to increase from the room temperature value by at least a factor of four when the specimen is maintained at a temperature of -120 degrees C on a liquid-nitrogen-cooled stage. A relationship derived between the critical dose, Ne, and the dose for optimum imaging, Nopt. Both Ne and Nopt depend, of course, upon the spatial frequency, or resolution. The derivation is valid only for the case in which all sources of noise other than quantum fluctuations are neglected. In this case, Nopt approximately equal to 2.5Ne. Finally, Nuclear Track Emulsion plates have been shown to be advantageous for recording high resolution electron diffraction patterns of small (1 micrometer 2) patches of crystalline biological materials.

Bacteriorhodopsins↗
PubMed1979

Prospects for extending the resolution limit of the electron microscope.

The factors that limit the performance of present-day high-resolution electron microscopes include the source brightness, the temporal coherence of the illumination, and phase contrast 'artefacts' due to spherical aberration and defocus. Further advancement of the instrumental performance might be accomplished in different ways, including the use of high coherence conditions combined with image restoration; the use of higher accelerating voltages; the use of aberration correctors; or the use of conditions for incoherent image formation. Current efforts in each of these directions are reviewed, from which it is evident that the combined use of higher voltages and improved coherence is the direction that is presently leading to the greatest degree of progress.

Electronics↗
PubMed1978

Molecular orientation of bacteriorhodopsin within the purple membrane of Halobacterium halobium.

The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.

Bacteriorhodopsins↗
PubMed1978

Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review.

When biological specimens are irradiated by the electron beam in the electron microscope, the specimen structure is damaged as a result of molecular excitation, ionization, and subsequent chemical reactions. The radiation damage that occurs in the normal process of electron microscopy is known to present severe limitations for imaging high resolution detail in biological specimens. The question of radiation damage at low temperatures has therefore been investigated with the view in mind of reducing somewhat the rate at which damage occurs. The radiation damage protection found for small molecule (anhydrous) organic compounds is generally rather limited or even non-existent. However, large molecular, hydrated materials show as much as a 10-fold reduction at low temperature in the rate at which radiation damage occurs, relative to the damage rate at room temperature. In the case of hydrated specimens, therefore, low temperature electron microscopy offers an important advantage as part of the overall effort required in obtaining high resolution images of complex biological structures.

Catalase↗
PubMed1977

Factors affecting high resolution fixed-beam transmission electron microscopy.

An experimental and theoretical characterization of a fixed-beam transmission electron microscope with a field emission gun has been made with regard to the factors of electron beam brightness, spatial and temporal coherence of the incident electrons, objective lens current fluctuation, mechanical stability, and specimen contamination. It has been found that mechanical stability and temporal coherence are the primary factors that prevent the contrast transfer function from extending to 2.0 A in our microscope. Different amorphous thin films have also been used in order to compare their suitability for testing the imaging capability of the microscope at atomic resolution.

Microscopy, Electron↗
PubMed1975

Development of methodology for low exposure, high resolution electron microscopy of biological specimens.

Specimen damage resulting from inelastic scattering is one of the factors that limits high-resolution electron microscopy of biological specimens. We have, therefore, sought to develop a method to record images of periodic objects at a reduced electron exposure in order to preserve high-resolution structural detail. The resulting image will tend increasingly to be a statistically noisy one, as the electron exposure is reduced to lower and lower values. Construction of a statistically defined image from such data is possible by spatial averaging of the electron signals from a large number of identical unit cells. In this paper, we have first investigated the theory pertaining to the attainable resolution as a function of the electron exposure, the magnification, and several other relevant parameters. In addition, we report experimental results obtained with a commercial image intensifier and with nuclear track photographic emulsion, both of which are highly sensitive recording devices. Usable images can be recorded and processed at exposures in the image plane as low as 10(-3) electron/micron2 (1.6 x 10(-14) coulomb/cm2).

Electrons↗
PubMed1975

Radiation damage in stained catalase at low temperature.

Radiation damage, which occurs in the beam of the electron microscope, has been studied in uranyl acetate stained crystals. Damage is observed in terms of the disappearance of higher orders of the electron diffraction pattern. In this study it has been found that the damage at very low specimen temperatures proceeds both more rapidly and to a greater final degree than is the case at room temperature.

Acetates↗
PubMed1975

Single atom image contrast: conventional dark-field and bright-field electron microscopy.

Single atom inage intensities are calculated for bright-field and various dark-field modes, including the ideal beam stop, a wire beam stop, tilted illumination and a displaced aperture. Comparisons of scattering amplitudes and elastic scattering cross-sections are made with different object potentials and scattering formulations. The image contrast for one mercury atom (Z equals 80) on a column of carbon atoms (Z equals 6) as the substrate is also discussed for both the bright-field and the various dark-field situations.

Carbon↗
PubMed1974

Electron diffraction of frozen, hydrated protein crystals.

High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique. The use of frozen specimens to maintain the hydration of complex biological structures has certain advantages over previously developed liquid hydration techniques.

Catalase↗