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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↗

PubMed1997

Flexibility and fine structure of smooth-muscle alpha-actinin.

The microfilament protein alpha-actinin exists as a dimer. The N-terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin-binding regions, while the C-terminal, larger parts consist of four spectrin-like repeats that interact to form a rod-like structure. To elucidate the fine structure of smooth-muscle alpha-actinin, we used energy-filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm x 4.8 nm in 0.05 M KCl and 32.6 nm x 4.4 nm in 0.15 M KCl. Both ends of the molecule displayed hook-like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N-terminal actin-binding regions. The rod-like center of the molecule revealed discrete globular masses which probably comprise the spectrin-like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod-like 53-kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm x 5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of alpha-actinin and suggest that effective actin-binding and cross-linking of alpha-actinin is based on the high flexibility of the terminal hooks.

Actinin↗

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↗

PubMed1997

Increase in extracellular calcium in the optic tectum of fish after optic nerve transection.

In this study the extracellular distribution of cytochemically generated calcium reaction product in the denervated optic tectum of a cichild fish (Oreochromis mossambicus) was investigated. The left optic nerve had been transected and the fish (5 per experimental condition) maintained for 2, 10 and 21 days. The amount of the calcium-containing precipitates was estimated using energy-filtering transmission electron microscopy (EFTEM) and image analysis. A special degeneration type of the optic terminals (neurofibrillar hypertrophy) was found which seems to be rare in other teleosts and was therefore chosen for quantification of the calcium deposits. These terminals are surrounded by astroglial processes and the calcium reaction product in the extracellular spaces between these glial processes and the terminals was measured and compared to normal optic terminals in nonoperated controls. A distinct and significant increase in the amount of calcium deposits was found 2 and 10 days after surgery which decreased to control levels after 21 days. This rise of deposits around the degenerating terminals was very local as arbitrarily selected extracellular spaces near these terminals showed values which were at the level of the nonoperated controls. Therefore, a transient and local increase in extracellular calcium precipitates was found after optic nerve transection which affected only the degenerating synapses.

Animals↗

PubMed1998

Possible Ca2+-dependent mechanism of apical outer hair cell modulation within the cochlea of the guinea pig.

Calcium ions were precipitated with potassium antimonate after injection of the inorganic calcium channel blocker MnCl2 or the inorganic potassium channel blockers BaCl2 or CsCl into the perilymph of the scala vestibuli of the guinea pig. The spatial distribution of the formed histochemical reaction products within the organ of Corti was studied by energy-filtering transmission-electron microscopy. Compared with untreated control ears, the number of the formed precipitates drastically increased at the extracellular side of the lamina reticularis after application of the various inorganic channel blockers. The apical side of the outer hair cells and the intervening Deiter cells were covered by a thick layer of calcium precipitates, whereas the number of histochemical reaction products was clearly reduced in the nearby acellular tectorial membrane. The high calcium content within the formed reaction products at the lamina reticularis could be demonstrated by elemental mapping and by electron energy-loss spectroscopy. To ascertain the alterations in the amounts of the calcium precipitates within the tectorial membrane after application of the various inorganic channel blockers, the precipitate densities were determined semiquantitatively by an image processing system and the values obtained compared with those of untreated control specimens. The observed histochemical results are in good agreement with published electrophysiological findings concerning the spatial distribution of ion channels located at the apical outer hair cell membrane. The detected alterations in the spatial distribution of calcium precipitates might correspond to calcium-dependent processes involved in outer hair cell modulation.

Animals↗

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↗

PubMedDate not supplied

Alteration of the calcium content in inner hair cells of the cochlea of the guinea pig after acute noise trauma with and without application of the organic calcium channel blocker diltiazem.

Calcium ions are known to be important to the process of signal transduction across the apical and basal sides of the inner hair cells. Calcium channel antagonists have been demonstrated by light microscopy to provide protection against acoustic trauma. To evaluate the protective effect of calcium channel blocker on the inner ear cells to noise exposure, the amount of the histochemical reaction products formed in the cytoplasm of the inner hair cells of the guinea pig after application of pyroantimonate was measured by an image processing system connected to an energy-filtering transmission electron microscope (EFTEM). Compared to untreated control specimens (experimental animal group I) the amount of precipitable calcium had clearly increased in the inner hair cells of noise-exposed cochleae 60 h after an acute noise trauma (group III). In addition, small electron- lucent areas could be identified in the cytoplasm of the hair cells probably representing damage to the cellular fine structure. When the calcium channel blocker diltiazem was administered without any additional noise exposure the calcium content was drastically reduced in the inner hair cells (group II), but when the antagonist was given before and after acute noise trauma (group IV), the calcium content in the inner hair cells was nearly compared to the amount determined in the untreated group of animals. The role of diltiazem is discussed in respect of tissue protection and to the maintenance of the calcium-dependent physiological processes in the inner hair cells during signal transduction.

Acute Disease↗

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↗

PubMed1997

Electron spectroscopic imaging analyses of the distribution of phosphorus in Balbiani ring granules and in the surrounding nucleoplasm.

The in situ distribution of phosphorus was studied in unstained ultrathin sections of salivary glands of Chironomus tentans and Ch. thummi larvae using elemental mapping by means of an energy-filtering transmission electron microscope. This distribution was related to the structures observed using contrast enhancement with inelastically scattered electrons at 250 eV. This procedure demonstrated that a phosphorus-containing fibril about 2 nm thick is the common substructure of the following nuclear ribonucleoprotein structural constituents: the Balbiani ring granules, their precursor fibrils seen at the sites of transcription, especially at the Balbiani rings, and the fibres traversing the pore of the nuclear envelope. These phosphorus-containing thin fibrils are sensitive to RNase. Thicker substructural features of the Balbiani ring granules, occurring as a curved ribbon on a dense particle, appear to be formed by the dense packing of the fine fibrils. The Balbiani ring granules located near the nuclear envelope are often linked to it by fine filaments.

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↗

PubMed1998

Combined characterization of composite tabular silver halide microcrystals by cryo-EFTEM/EELS and cryo-STEM/EDX techniques.

The combination of cryo-energy filtering transmission electron microscopy (EFTEM)/electron spectroscopic diffraction (ESD)/electron energy-loss spectroscopy (EELS) and cryo-energy-dispersive X-ray (EDX) analysis in the scanning transmission (STEM) and scanning (SEM) modes was applied for the characterization of composite tabular Ag(Br,I) microcrystals. A low-loss fine structure in EEL spectra between 4 and 26 eV was attributed to excitons and plasmons possibly superimposed with interband transitions and many-electron effects. The contrast tuning under the energy-filtering in the low-loss region was used to image the crystal morphology, defect structure (random dislocations and ¿111¿ stacking faults) and bend and edge contours as well as electron excitations in the microcrystals. Sharp extra reflections at commensurate positions in between the main Bragg reflections and diffuse honeycomb contours in ESD patterns of the microcrystals taken near the [111] zone were assigned to the number of defects in the shell region parallel to the grain edges and polyhedral clusters of interstitial silver cations, respectively. The imaginary part of the energy-loss function, Im (-1/epsilon), and the real and imaginary parts, epsilon1 and epsilon2, of the dielectric permittivity were determined by means of a Kramers-Kronig analysis. An assignment of exciton peaks based on calculations of electronic band structure of silver bromide is proposed. Inner-shell excitation bands of silver halide were detected in line with EDX-analyses. The energy-loss near-edge structure (ELNES) of the AgM4,5-edge governed by spin-orbital splitting between the 3d3/2- and 3d5/2-states has been evaluated. Combined silver and halide distributions were obtained by a three-window method (EFTEM) and by EDX/STEM including area mapping and line profiling of iodide.

Bromides↗

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↗

PubMed2000

Valence state mapping of cobalt and manganese using near-edge fine structures

The properties of transition metal oxides are related to the presence of elements with mixed valences. The spectroscopy analysis of the valence states is feasible experimentally, but a spatial mapping of valence states of transition metal elements is a challenge to existing microscopy techniques. In this paper, with the use of valence state information provided by the white lines and near-edge fine structures observed using the electron energy-loss spectroscopy (EELS) in a transmission electron microscope (TEM), a novel experimental approach is demonstrated to map the valence state distributions of Mn and Co using the ratio of white lines in the energy-filtered TEM. The valence state map is almost independent of specimen thickness in the thickness range adequate for quantitative EELS microanalysis. An optimum spatial resolution of approximately 2 nm has been achieved for a two-phase Co oxides.

Journal Article↗

PubMed1999

Quantification of total calcium in terminal cisternae of skinned muscle fibers by imaging electron energy-loss spectroscopy.

Skinned muscle fibers are ideal model preparations for the investigation of Ca2+ -regulatory mechanisms. Their internal ionic milieu can be easily controlled and distinct physiological states are well defined. We have measured the total Ca content in the terminal cisternae of such preparations using imaging electron energy-loss spectroscopy (Image-EELS) as a new approach for quantification of sub-cellular element distributions. Murine muscle fibers submitted to a standardized calcium-loading procedure were cryo-fixed with a combined solution exchanger/plunge freezing device. Energy-filtered image series were recorded from ultrathin freeze-dried cryosections of samples immobilized in either relaxed or caffeine-contracted state. From these image series, electron energy-loss spectra were extracted by digital image-processing and quantitatively processed by multiple-least-squares-fitting with reference spectra. The calculated fit coefficients were converted to Ca-concentrations by a calibration obtained from Ca-standards. Total Ca-contents in the terminal cisternae of skinned skeletal muscle fibers decreased upon caffeine-induced Ca-release from 123+/-159 (+/-11) to 73+/-102 (+/-8) mmol/kg d.w. (weighted mean +/- SD (+/-SEM)).

Animals↗