SEARCH · PubMed Health

Results for “Microscopy, Energy-Filtering Transmission Electron”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1986

Elemental imaging and resolution in energy-filtered conventional electron microscopy.

Energy-filtered transmission electron microscopy (EFEM) was used to image the distributions of uranium and carbon in uranyl acetate stained catalase crystals. The spatial resolution obtained from inelastic C K-edge and U O4,5-edge images, determined from the highest-order reflection in the computed diffraction pattern, was 3.4 nm for both carbon and uranium. The resolution limit imposed by the delocalization of inelastic scattering was estimated from cross-section measurements to be 0.6 nm for U and 0.2 nm for C. Considering both delocalization and the effects of microscope aberrations, for an objective lens chromatic aberration coefficient of 2.8 mm and 10 eV energy window, the calculated resolutions are 2.0 nm for C and 1.2 nm for U. The effects of plural inelastic and elastic-inelastic scattering were sufficiently large to show crystalline structure in unprocessed pre-edge inelastic images. Previously suggested methods for eliminating these artifacts were applied to obtain the compositional information in the catalase EFEM images.

Carbon↗

PubMed1996

Imaging of thick sections of nervous tissue with energy-filtering transmission electron microscopy.

Electron microscopy of nervous tissue requires on the one hand nanometre resolution for the analysis of fine structures of nerve cell contacts, for instance synaptic vesicles, synaptic membranes and associated organelles. On the other hand, the visualization of the three-dimensional organization of nervous tissue on the level of dendrites and neurites is essential for the understanding of neuronal integration and also for a stereological evaluation of quantitative parameters such as size and shape of synaptic contact zones, number and distribution of synaptic vesicles, organization of cytoskeleton and distribution of organelles like mitochondria and endoplasmic reticulum. Therefore, it is necessary to have access to the fine structure and to the spatial organization within one sample. Energy-filtering transmission electron microscopy (EFTEM) enables the imaging of sections up to 1 micron thickness with a high resolution because the chromatic error due to inelastic scattering is avoided by selecting electrons within a restricted energy-loss range for imaging. The contrast depends strongly upon the section thickness, the selected energy range and the composition of the sample, so that optimum imaging conditions can be found for each object. Different staining protocols enable either a high global contrast or a selective staining of peculiar tissue properties. The three-dimensional organization can be visualized with stereopairs or with extended tilt series, which shows that characteristic structures as the synaptic junctions are detectable only within a narrow range of orientations to the electron beam. This is especially important for quantitative approaches with stereological tools which profit generally from the fact that a wide range of section thickness is available with EFTEM. EFTEM is therefore a powerful tool for the imaging of thick sections of biological materials with attractive possibilities of contrast tuning and advantages for stereological quantifications. The main benefit is the rapid and effective visualization of the three-dimensional organization of cells and tissues.

Animals↗

PubMed1982

Energy-filtered transmission electron microscopy of ferritin.

The focusing properties of a magnetic-sector spectrometer are shown to be suitable for forming high-spatial-resolution, energy-filtered transmission electron microscope images. Filtered images of ferritin molecules by using electrons scattered from the characteristic iron M2,3 and carbon K absorption edges clearly distinguish the 75-A iron core and 120-A protein shell. The minimum detectable mass is estimated to be 0.84 X 10(-20) g for Fe for an electron dose of 18 C/cm2 and 99% confidence.

Ferritins↗

PubMed1994

Energy-filtering transmission electron microscopy as a tool for structural and compositional analysis of isolated ferritin particles.

Structural and compositional analysis of isolated horse-spleen ferritin particles was performed by energy filtering transmission electron microscopy (EFTEM). Ferritin particles were collected in ultrathin (2 nm thick) chromium films and analyzed without any additional stain by electron energy-loss spectroscopy (EELS) for iron and carbon and by electron-spectroscopic imaging (ESI) for carbon. The ultrastructure of the proteinaceous shell of the ferritin particle, as obtained by the carbon net-intensity electron spectroscopical and carbon concentration-distribution images, was qualitatively compared to the structure as acquired by a negative-staining procedure. Quantitative analysis of the number of carbon atoms in the ferritin-shell proteins was carried out through an ESI-acquisition protocol and processing procedure with calibrated attenuation filters in the optical path to the TV camera. This procedure included images acquired with calibrated attenuation filters for the compensation of shading and the non-linear performance of the TV camera used in the analytical part of the procedure. A new ¿ESI-Spectra¿ program is proposed that allows element-related spectra to be generated at any place and with any frame size in a contrast-sensitive or other type of image present on the computer monitor screen.

Animals↗

PubMed1993

Analysis of early hard tissue formation in dentine by energy dispersive X-ray microanalysis and energy-filtering transmission electron microscopy.

Thin cryosections and sections of embedded tissue were prepared from dentine of cryofixed rat incisors. Energy dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have been applied to study the calcium and phosphorus distribution in predentine of these incisors. A small enrichment of calcium and phosphorus was found in the predentine zone near the dentine border. Element distributions were correlated with analyses of the early crystal formation in dentine. These investigations were carried out by parallel applications of electron spectroscopic diffraction (ESD) and electron spectroscopic imaging (ESI) using zero-loss filtering. It was found that the earliest crystal formations already showed the lattice of the hexagonal mineral apatite. They form parallelly arranged chains of dots which coalesce rapidly to form "needle-like" crystallites along the collagen microfibrils.

Animals↗

PubMed1993

Characteristic and differential calcium signals from cell structures of the root cap detected by energy-filtering electron microscopy (EELS/ESI).

Characteristic calcium signals were analyzed in structures of three cell types of the root cap of cress: statocytes, meristematic cells and secretion cells. Twenty-four hour-old roots were fixed with glutaraldehyde (postfixed with osmiate/potassium bichromate) or with potassium permanganate. No visible precipitates were formed, but calcium signals typical for different cell structures could be detected by means of energy-filtering transmission electron microscopy (EELS/ESI). In statocytes, calcium signals were recorded from the plasma membrane, the membranes of the endoplasmic reticulum, the amyloplast envelope and the mitochondrial membranes. In contrast to the excitable statocytes, the two other, non-excitable cell types, meristematic and secretion cells, exhibited much lower intensities of the calcium signals recorded from the same membrane systems. The revealed characteristic calcium-related properties of the different membrane systems may be related to the special function of statocytes, namely transduction of the gravity stimulus. In all three cell types, additional calcium signals were recorded from cell structures with well known calcium contents, i.e., mitochondrial granules, starch grains and cell walls. For the first time, clear calcium signals were detected from the lipid bodies which are mobilized during the developmental stage of the examined roots. It is supposed that free fatty acids and lipases are the binding sites for calcium. The reliability of the applied method is especially proven by comparison of the electron microscopic images from lipid bodies according to the two different fixation methods. After glutaraldehyde fixation followed by osmiate/potassium bichromate postfixation, the lipid bodies were well fixed and appeared homogeneously grey with homogeneous calcium signals. However, due to potassium permanganate fixation the lipid bodies were only partly fixed; they had an electron-lucid core, from which the lipids are lost during the dehydration procedure, without calcium signals and an electron-dense border zone, which is a reaction product of potassium permanganate with triacylglycerols, with calcium signals.

Calcium↗

PubMed1995

Comparative methodological investigations on the cytochemical localization of calcium in brain and inner ear of cichlid fish.

Four different methods for calcium precipitation are compared in the optic tectum and the inner ear of the cichid fish, Oreochromis mossambicus. Several parameters are investigated concerning their influences on the reaction product. Three procedures (bichromate, fluoride, and oxalate-pyroantimonate) produce fine-grained deposits, often flocculent in the latter method. The fourth method (potassium-pyroantimonate) generates predominantly coarse-grained reaction product. The calcium content of the deposits is always proven with energy-filtering transmission electron microscopy (EFTEM). In both tissues fine-grained reaction product is found in endoplasmic reticulum and synaptic vesicles, and in addition in some mitochondria and at the cytoskeleton. The coarse-grained deposits of the potassium-pyroantimonate method have a more unspecific distribution. This is the only method which produces extracellular deposits in the inner ear, whereas in the optic tectum extracellular precipitates are always present except with the oxalate-pyroantimonate procedure. Two factors have an influence on the reaction product: the duration of fixation and the type of resin. The prolongation of the fixation time up to 24 hours leads to an increase of the reaction product, which also becomes coarse-grained. These observations are corroborated by quantification with image analysis. Furthermore the use of an epoxy resin compared to acrylic resins decreases the amount of reaction product produced. We show that the application of several methods is meaningful in order to understand the calcium properties of the investigated tissue, but it is necessary to optimize a certain method for a given tissue.

Animals↗

PubMed1992

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue.

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.

Animals↗

PubMed1994

Image-EELS: a synthesis of energy-loss analysis and imaging.

Two different modes of energy-filtering transmission electron microscopy (EFTEM) are often used for element microanalysis: electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). A new approach was developed which we call Image-EELS. This procedure was realized with the commercially available standard equipment of the energy-filtering transmission microscope CEM 902 (Zeiss, Germany). A series of energy-filtered images is recorded with ESI at many different energy losses. In a second step the intensity of selected objects is measured for each energy loss and plotted as a function of the energy loss, that means as an EELS spectrum. This method increases the sensitivity of EELS analysis, especially for very small and irregular objects, because the lateral resolution is enhanced and the noise is suppressed by the integration of many pixels belonging to one type of object. Many spectra can be calculated from one image series, enabling the comparison of spectra from different objects. Selected images from the series can be used for ESI elemental mapping, so that errors and limits in the different mapping procedures can be detected. Image-EELS is a synthesis of EELS and ESI and as such it constitutes a considerable progress for element microanalysis with EFTEM, not only for biological objects.

Animals↗

PubMed1993

Influence of seasonal adaptation on the distribution of cytochemically detectable endogenous calcium in synaptic terminals of the optic tectum of crucian carp (Carassius carassius L.).

Crucian carps were seasonally adapted to high (23 degrees C) or low (4 degrees C) temperatures and synaptic regions in the optic tectum were analyzed by means of energy-filtering transmission electron microscopy (EFTEM). The amount of cytochemically detectable endogenous calcium in synaptic clefts was quantified by means of image analysis and the amount of glycogen granules in synaptic regions was assessed. In cold-adapted fish, glycogen is increased, especially in glial cells, while the calcium-containing precipitates are reduced. This is interpreted as a characteristic feature of inactive synapses, in which glycogen accumulates and in which the calcium turnover is slowed down. Short-term thermal stress did not influence the amount of calcium or glycogen.

Acclimatization↗

PubMed1996

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging.

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron-specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 microns) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.

Animals↗

PubMed1992

Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus.

We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.

Computer Simulation↗

PubMed1993

Applications of a post-column imaging filter in biology and materials science.

We have developed an energy-selecting imaging filter which can be attached to most transmission electron microscopes, and is capable of operating at primary energies of up to 400 keV. We review the filter's design and operation, and illustrate them with several applications including imaging of thick biological materials, energy-filtered diffraction, energy-filtered high-resolution imaging, and elemental mapping. We conclude the paper with a discussion of the areas of transmission electron microscopy in which the filter is likely to make especially significant contributions.

Equipment Design↗

PubMed1990

Quantitative energy-filtered image analysis in cytochemistry. I. Morphometric analysis of contrast-related images.

A combination of energy-filtered electron microscopy (EFEM) and an image-analyzing system (IBAS/2000) is used for morphometric analyses of cells and (reaction) products. Image contrast is objectively established and segmentation is based upon intrinsic contrasts, in ultrathin sections. Cross-sectioned platinum-stained erythrocytes are used as a model to determine optimal conditions for constant measuring results for contrast, area and perimeter. Results are related to changes in: (1) the objective-lens diaphragm diameter, (2) three most frequently used contrast modes obtainable by electron spectroscopical imaging (ESI) in a Zeiss EM 902 transmission electron microscope (e.g., global, zero loss (or deltaE - 0 eV) and deltaE = 250 eV), and (3) the number of image integrations (1-250X) acquired by real-time video. A thresholding procedure is proposed for objective segmentation of such contrast-related images and applied to measure the area fraction of nuclear chromatin and the diameter of nominal 1 nm colloidal gold particles.

Animals↗

PubMed1996

Probing nuclear ultrastructure by electron spectroscopic imaging.

Mammalian nuclei are complex organelles containing many functionally distinct nucleoprotein and protein particles in the size range 20-30 nm. This complexity hinders the study of structure-function relationships within the mammalian nucleus. Element-specific mapping using the energy-filtered transmission electron microscope can provide novel information on protein and nucleic acid density within structures, facilitating the identification of biochemical heterogeneity within morphologically similar structures. We demonstrate that imaging phosphorus, nitrogen and carbon can be useful in the characterization of protein and nucleoprotein structures within the nucleus. Additionally, electron spectroscopic imaging (ESI) may be used to map the distribution of strains relative to unstained material when biochemical-specific staining protocols, such as EDTA-regressive staining of RNA with uranyl acetate, are used. Relative mass may also be determined from ESI images and can be combined with elemental information further to distinguish biological constituents. Using this approach, heterochromatin was found to be variable in nucleic acid content although the morphology appeared relatively homogenous. ESI shows substantial promise for the investigation of structure-function relationships in biological specimens.

Animals↗

PubMed1996

Quantitative electron spectroscopic imaging in bio-medicine: evaluation and application.

Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components. The concentration measurement scale is approximately linear up to a relative section thickness of approximately equal to 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion. Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.

Ferritins↗

PubMed1990

EELS elemental mapping with unconventional methods. II. Applications to biological specimens.

This article presents two applications of image analysis and processing using the unconventional methods described in the companion paper (part I). Both the information analysis via relative entropy measurement and mapping and the factorial analysis of correspondence are demonstrated to be valuable tools for building an elemental map from a set of noisy energy-filtered images recorded in an analytical transmission electron microscope. Although the only technique considered here is electron energy loss spectroscopy, there is no doubt that such methods can be applied to a wide variety of similar problems: only a reduced number of underlying hypotheses are needed.

Algorithms↗

PubMed1991

Contrast in the electron spectroscopic imaging mode of a TEM. IV. Thick specimens imaged by the most-probable energy loss.

When the zero-loss transmission falls below 10(-3) for biological sections of mass-thickness greater than 70 micrograms/cm2, the energy window in the electron spectroscopic imaging (ESI) mode of an energy-filtering electron microscope (EFEM) can be shifted to the most-probable energy loss of the electron energy-loss spectrum. This enables mass-thicknesses up to 150 micrograms/cm2 or thicknesses of 1.5 microns to be examined. Electron energy-loss spectra of thick carbon films calculated by a Fourier method agree with experimental spectra. Measurements of the electron energy-loss spectroscopy and ESI image intensities with an additional platinum film confirm a scattering model for the calculation of the image intensity. This model considers the angular broadening at the most-probable energy loss by introducing an effective illumination aperture of the order of the full-width at half-maximum of the angular distribution.

Fourier Analysis↗