Observation of biological macromolecules using various electron microscopic methods

1978 ◽  
Vol 36 (2) ◽  
pp. 170-171
Author(s):  
Mitsuo Ohtsuki ◽  
Michael Sogard
Keyword(s):  
Electron Microscope ◽  
Phase Contrast ◽  
Image Interpretation ◽  
Density Lipoprotein ◽  
Biological Macromolecules ◽  
Contrast Effects ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Structural Investigations ◽  
Staining Techniques

Structural investigations of biological macromolecules commonly employ CTEM with negative staining techniques. Difficulties in valid image interpretation arise, however, due to problems such as variability in thickness and degree of penetration of the staining agent, noise from the supporting film, and artifacts from defocus phase contrast effects. In order to determine the effects of these variables on biological structure, as seen by the electron microscope, negative stained macromolecules of high density lipoprotein-3 (HDL3) from human serum were analyzed with both CTEM and STEM, and results were then compared with CTEM micrographs of freeze-etched HDL3. In addition, we altered the structure of this molecule by digesting away its phospholipid component with phospholipase A2 and look for consistent changes in structure.

scholarly journals An Electron Microscopic Study of the Changes in Platelets during Viscous Metamorphosis

Blood ◽  
1962 ◽  
Vol 20 (5) ◽  
pp. 566-580 ◽  
Author(s):  
P. A. CASTALDI ◽  
B. G. FIRKIN ◽  
P. M. BLACKWELL ◽  
K. I. CLIFFORD
Keyword(s):  
Electron Microscope ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Light Microscope ◽  
Phase Contrast ◽  
Intact Cell ◽  
Complete Loss ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Sectioned Material

Abstract Viscous metamorphosis of platelets has been studied with the light microscope, and ultra-thin sections have been prepared at progressive stages for examination in the electron microscope. The phase contrast light microscope reveals rapid aggregation and distortion of platelets and gives the impression of their fusion into structureless aggregates during viscous metamorphosis. Sectioned material collected during viscous metamorphosis of platelets and examined in the electron microscope reveals a remarkable degree of retention of structure in a majority of the platelets. All become deficient in granules and devoid of vesicular spaces, but most retain intact cell membranes, and the structureless masses seen with the light microscope are found to consist of densely aggregated platelets. Fusion and complete loss of identity occurs in the minority. The retracted clot was found to contain densely aggregated, distorted and elongated platelets, empty of granules and intimately related to fibrin particles.

Cryoelectron microscopy and image reconstruction of spherical viruses with spot scan and FEG technologies

1995 ◽  
Vol 53 ◽  
pp. 1086-1087
Author(s):  
N. H. Olson ◽  
U. Lücken ◽  
S. B. Walker ◽  
M. T. Otten ◽  
T. S. Baker
Keyword(s):  
Electron Microscope ◽  
Transfer Function ◽  
Field Emission ◽  
Phase Contrast ◽  
Atomic Resolution ◽  
Bright Spot ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Transmission Electron ◽  
Chlorotic Mottle

The field emission gun electron microscope (FEG) is a tool that has the potential to achieve near atomic resolution information of biological macromolecules. The FEG provides a beam with higher spatial and temporal coherence and a better phase contrast transfer function than do microscopes with either tungsten or LaB6 filaments. The FEG is also ideal for spot scan imaging applications because it can produce a small, coherent and very bright spot. In spot scan mode the specimen is exposed to an array of nonoverlapping spots rather man a flood beam. This significantly reduces beam-induced specimen drift.Frozen-hydrated samples of cowpea chlorotic mottle (CCMV, Fig. 1A) and cowpea severe mosaic virus (CPSMV, Fig. IB) were examined on a Philips CM12 transmission electron microscope equipped with a standard LaB6 gun and on a Philips CM200 equipped with a field emission gun, respectively. The CM12 was operated at 120kV and was externally controlled by means of a spot scan imaging program which produced a series of 250 nm diameter spots on Kodak SO-163 sheet film.

Correlation of cryo-electron microscopic and x-ray data and compensation of the contrast transfer function

1992 ◽  
Vol 50 (2) ◽  
pp. 996-997
Author(s):  
R. Holland Cheng
Keyword(s):  
Transfer Function ◽  
Phase Contrast ◽  
Objective Lens ◽  
Biological Macromolecules ◽  
Electron Microscopic ◽  
Contrast Transfer Function ◽  
Image Reconstruction Techniques ◽  
X Ray ◽  
Mass Distributions ◽  
Spatial Frequencies

Cryo-electron microscopy (cryoEM) along with image reconstruction techniques can produce vivid images of biological macromolecules in their “native” state, although objective interpretation of these images is influenced by the fact that the contribution of phase contrast greatly exceeds that of amplitude contrast in such weakly scattering objects. The microscope contrast transfer function (CTF), which is strongly dependent on the defocus level of objective lens, modulates images of the object density distribution as a function of spatial frequency. Compensation for the effects of phase contrast transfer is important because underweighting of the low spatial frequencies usually causes difficulties in evaluating absolute mass distributions in objects.Correct compensation for the CTF is difficult to achieve. This is due, in part, to ambiguities in measuring the exact defocus level in noisy micrographs, and in knowing the relative contributions of amplitude and phase contrast, beam coherence, and inelastic scattering. The availability of atomic resolution determinations for a few viruses allows one to determine empirically how to correct the cryoEM images to best fit the x-ray data.

A Reproducible Selective Stain for Cardiac Sarcoplasmic Reticulum

1974 ◽  
Vol 32 ◽  
pp. 214-215
Author(s):  
R. A. Waugh ◽  
J. R. Sommer
Keyword(s):  
Complex System ◽  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Transmission Electron

Cardiac sarcoplasmic reticulum (SR) is a complex system of intracellular tubules that, due to their small size and juxtaposition to such electron-dense structures as mitochondria and myofibrils, are often inconspicuous in conventionally prepared electron microscopic material. This study reports a method with which the SR is selectively “stained” which facilitates visualizationwith the transmission electron microscope.

Analysis of STEM micrographs of thick objects

1978 ◽  
Vol 36 (2) ◽  
pp. 106-107
Author(s):  
S. Golladay
Keyword(s):  
Inelastic Scattering ◽  
Multiple Scattering ◽  
Mass Distribution ◽  
Cross Sections ◽  
Phase Contrast ◽  
Image Point ◽  
Contrast Effects ◽  
Total Cross Sections ◽  
Elastic And Inelastic Scattering

The theory of multiple scattering has been worked out by Groves and comparisons have been made between predicted and observed signals for thick specimens observed in a STEM under conditions where phase contrast effects are unimportant. Independent measurements of the collection efficiencies of the two STEM detectors, calculations of the ratio σe/σi = R, where σe, σi are the total cross sections for elastic and inelastic scattering respectively, and a model of the unknown mass distribution are needed for these comparisons. In this paper an extension of this work will be described which allows the determination of the required efficiencies, R, and the unknown mass distribution from the data without additional measurements or models. Essential to the analysis is the fact that in a STEM two or more signal measurements can be made simultaneously at each image point.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

The Imaging of Crystal Structures and Crystal Defects

1978 ◽  
Vol 36 (3) ◽  
pp. 207-217
Author(s):  
J.M. Cowley
Keyword(s):  
Electron Microscope ◽  
Crystal Structures ◽  
Phase Contrast ◽  
Crystal Defects ◽  
Atom Density ◽  
Lack Of Information ◽  
Spatial Frequencies

The problem of "understandinq" electron microscope imaqes becomes more acute as the resolution is improved. The naive interpretation of an imaqe as representinq the projection of an atom density becomes less and less appropriate. We are increasinqly forced to face the complexities of coherent imaqinq of what are essentially phase objects. Most electron microscopists are now aware that, for very thin weakly scatterinq objects such as thin unstained bioloqical specimens, hiqh resolution imaqes are best obtained near the optimum defocus, as prescribed by Scherzer, where the phase contrast imaqe qives a qood representation of the projected potential, apart from a lack of information on the lower spatial frequencies. But phase contrast imaqinq is never simple except in idealized limitinq cases.

Scanning Electron Microscopic Study of the Cell Surface

1969 ◽  
Vol 27 ◽  
pp. 36-37 ◽  
Author(s):  
Toichiro Kuwabara
Keyword(s):  
Tissue Fluid ◽  
Biological Application ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Surface Appearance ◽  
Minute Amount ◽  
Scanning Electron Microscopic Study ◽  
Scanning Electron Microscopic ◽  
True Structure ◽  
Scanning Electron

Although scanning electron microscopy has a great potential in biological application, there are certain limitations in visualization of the biological structure. Satisfactory techniques to demonstrate natural surfaces of the tissue and the cell have been reported by several investigators. However, it is commonly found that the surface cell membrane is covered with a minute amount of mucin, secretory substance or tissue fluid as physiological, pathological or artefactual condition. These substances give a false surface appearance, especially when the tissue is fixed with strong fixatives. It seems important to remove these coating substances from the surface of the cell for demonstration of the true structure.

Freeze-Fracture Replication in the Ultrastructure of Blue-Green Algae

1967 ◽  
Vol 25 ◽  
pp. 74-75
Author(s):  
L. V. Leak
Keyword(s):  
Cell Wall ◽  
Electron Microscope ◽  
Green Algae ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Electron Microscopic ◽  
Photosynthetic Membranes ◽  
Blue Green Algae ◽  
Cell Biol ◽  
Cellular Components

Electron microscopic observations of freeze-fracture replicas of Anabaena cells obtained by the procedures described by Bullivant and Ames (J. Cell Biol., 1966) indicate that the frozen cells are fractured in many different planes. This fracturing or cleaving along various planes allows one to gain a three dimensional relation of the cellular components as a result of such a manipulation. When replicas that are obtained by the freeze-fracture method are observed in the electron microscope, cross fractures of the cell wall and membranes that comprise the photosynthetic lamellae are apparent as demonstrated in Figures 1 & 2.A large portion of the Anabaena cell is composed of undulating layers of cytoplasm that are bounded by unit membranes that comprise the photosynthetic membranes. The adjoining layers of cytoplasm are closely apposed to each other to form the photosynthetic lamellae. Occassionally the adjacent layers of cytoplasm are separated by an interspace that may vary in widths of up to several 100 mu to form intralamellar vesicles.

A scanning electron microscopic study on dissolving process of cholesterol gallstones by chenodeoxycholic acid (CDCA)

1978 ◽  
Vol 36 (2) ◽  
pp. 522-523
Author(s):  
T. Kanetaka ◽  
M. Cho ◽  
S. Kawamura ◽  
T. Sado ◽  
K. Hara
Keyword(s):  
Electron Microscope ◽  
Chenodeoxycholic Acid ◽  
Outer Surface ◽  
Electron Microscopic ◽  
Cholesterol Gallstones ◽  
The Core ◽  
Scanning Electron Microscopic Study ◽  
Scanning Electron Microscopic ◽  
Acceleration Voltage ◽  
Scanning Electron

The authors have investigated the dissolution process of human cholesterol gallstones using a scanning electron microscope(SEM). This study was carried out by comparing control gallstones incubated in beagle bile with gallstones obtained from patients who were treated with chenodeoxycholic acid(CDCA).The cholesterol gallstones for this study were obtained from 14 patients. Three control patients were treated without CDCA and eleven patients were treated with CDCA 300-600 mg/day for periods ranging from four to twenty five months. It was confirmed through chemical analysis that these gallstones contained more than 80% cholesterol in both the outer surface and the core.The specimen were obtained from the outer surface and the core of the gallstones. Each specimen was attached to alminum sheet and coated with carbon to 100Å thickness. The SEM observation was made by Hitachi S-550 with 20 kV acceleration voltage and with 60-20, 000X magnification.

Sign in / Sign up

Export Citation Format

Share Document