Energy-filtering transmission electron microscopy (EFTEM) in the elemental analysis of pseudoexfoliative material.

PURPOSE: To obtain more information on the basic nature of the pathological matrix product accumulating in pseudoexfoliation (PEX) syndrome by analyzing its elemental composition at the subcellular level. METHODS: Energy-filtering transmission electron microscopy (EFTEM), combining the two microanalytical techniques of electron spectroscopic imaging (ESI) and energy-loss spectroscopy (EELS), were performed on ultrathin sections of lens specimens with PEX syndrome using a transmission electron microscope equipped with an integrated electron energy filter. EFTEM is based on inner shell ionization of elements present in the sample giving rise to characteristic signals in well-defined energy-loss regions. The EEL-spectra, demonstrating the presence of a particular element by its specific electron energy-loss edge, were recorded with an integrated scintillator-photomultiplier-system. ESI generated graphic images of elemental localization in the sections after a process of background correction with an IBAS image analysis program. Energy-dispersive X-ray (EDX) analysis of PEX deposits on hydrated lenses was conducted by variable pressure scanning electron microscopy. RESULTS: The ESI element distribution images of both intracapsular and supracapsular PEX material displayed high signals for nitrogen, sulfur, calcium, chlorine, and zinc in clear association with the PEX fibrils. The corresponding EEL-spectra confirmed the data obtained by ESI and showed the presence of the element-specific energy-loss edges. The presence of these elements in PEX fibrils was further confirmed by EDX analysis. No specific signals were obtained for phosphorus, oxygen, or aluminum. CONCLUSIONS: This study demonstrates the presence of nitrogen, sulfur, chlorine, zinc, and calcium both in mature and in aggregating PEX fibrils of the lens capsule. EFTEM proved to be a highly sensitive method for the microanalytical study of biological material with unknown composition, such as PEX material, at the subcellular level.

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.

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.

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.

Correlation of altered gravity and cytochrome oxidase activity in the developing fish brain.

The mitochondrial enzyme, cytochrome oxidase, was localized cytochemically in the nucleus magnocellularis, a primary relay nucleus of vestibular information within the area octavolateralis in the fish brain. Cichlid fish larvae were analyzed after long-term exposure (9 days) to altered gravity situations: increased acceleration in a centrifuge (3 g) and near weightlessness during space flight. Controls (1 g) were reared under identical conditions in the centrifuge but without rotation on earth or with an acceleration resulting in gravity of 1 g in space shuttle. Quantification of highly reactive mitochondria reveals a correlation of gravity and cytochrome oxidase activity: low enzyme activity in respect to 1 g controls under near weightlessness conditions and an increased activity after hyper-g exposure in a centrifuge. This gravity effect on the energy metabolism of vestibular nuclei of developing cichild fish seems to reflect adaptational processes in response to gravity stimulation.

Comparative electronmicroscopical investigations on the influences of altered gravity on cytochrome oxidase in the inner ear of fish: a spaceflight study.

The regional metabolic activity in the otolithic sensory epithelia of the inner ear of a cichlid fish (Oreochromis mossambicus) was investigated on light- and electronmicroscopical level using the cytochemical method for detection of cytochrome oxidase activity. In adult animals a characteristic distribution of mitochondria with high enzyme activity was found in sensory and non-sensory cells of otolithic sensory epithelia, which was correlated with regions with a high energy demand. These findings were the basis for studies on the influence of long-term altered gravity conditions in developing larvae: hypogravity (10(-4) g in spaceflight), normal gravity (1 g in a centrifuge in space and 1 g on earth) and hypergravity (3 g in a laboratory centrifuge). Cytochrome oxidase activity was quantified in different parts of the sensory hair cell synapse in the vestibular sensory epithelia utricle and saccule: apical and basal cytoplasm, postsynaptic area of the afferent synapse and presynaptic region of the efferent synapse. Our results show that the energy metabolism of utricle, but not of saccule is decreased after microgravity exposure during the 2nd German Spacelab Mission D-2. However, a general effect of the spaceflight is detectable in both sensory epithelia. Long-term exposure to increased acceleration (3 g) had no effects on cytochrome oxidase activity in inner ear sensory epithelia.

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.

Localization of NADPH-diaphorase activity in the salt gland of the saltwater-acclimated Pekin duck.

Exocrine secretion of the avian salt gland is controlled by the autonomic nervous system. NADPH-diaphorase histochemistry was employed at the light and electron microscopic level to provide the morphological basis for a putative nitrergic regulation of salt gland function. NADPH-diaphorase staining was localized in two cell populations of the parasympathetic secretory ganglion at high cell density and equal distribution throughout the ganglionic mass. In addition, salt gland-intrinsic neurons, arranged in small clusters and associated with major nitrergic fiber bundles, proved to be NADPH-diaphorase positive. These postganglionic nerve fibers innervated the secretory parenchyma in close proximity to the basal membrane of single secretory tubules as well as arterioles. The findings suggest participation of the nitrergic pathway in the autonomic control of avian salt gland function.

Effect of light stimulation on the cytochemical localization of cytochrome C oxidase in photoreceptor cells of cichlid fish.

Cytochrome oxidase activity was evaluated cytochemically in rod photoreceptor cells in the retina of the cichlid fish Oreochromis mossambicus after different stimulation protocols. The enzyme activity was assessed semiquantitatively by estimating the volume ratio of mitochondria classified according to the intensity of enzyme reactivity. Dark adaptation for 5 hr induced an increase of cytochrome oxidase activity both in vivo and in vitro, i.e., in isolated retinas. Short-term illumination (1 hr) of isolated retinas adapted previously in vivo to darkness caused a significant decrease of enzyme activity, whereas short-term darkening after in vivo light adaptation had no effect. Chemical stimulation for 15 min with increased K+ concentration (20 mM) reduced the enzyme activity, i.e., chemical depolarization did not have the same effect as depolarization induced by darkening. Significant changes in cytochrome oxidase activity were apparent within 1 hr of stimulation, so that this method for analysis of neuronal activity can be applied even in short-term experiments.

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.

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.

Effects of development and altered gravity conditions on cytochrome oxidase activity in a vestibular nucleus of the larval teleost brain: a quantitative electronmicroscopical study.

The mitochondrial enzyme, cytochrome oxidase, was localized cytochemically in the nucleus magnocellularis, a primary relay nucleus of vestibular information within the area octavolateralis in the fish brain. Larvae of the cichlid fish Oreochromis mossambicus were analyzed at different developmental stages (4, 10, and 35 days post-hatching) and after long-term exposure (8 days) to increased gravity (2-4 g). Quantification of highly reactive, moderately reactive, and nonreactive mitochondria reveals differences in the cytochrome oxidase activity of various cellular structures, for example, perikarya of neurons, presynaptic terminals, and myelinated and nonmyelinated cell profiles. Cytochrome oxidase activity in the mitochondria of neuronal perikarya increases during development which parallels the differentiation of the area octavolateralis. This possibly reflects the increasing energy demand during maturation and innervation of the magnocellular nucleus. Hyper-g-exposure of the larvae for 8 days (centrifuge) caused a further augmentation of cytochrome oxidase activity in the perikarya within the nucleus magnocellularis. This may reflect an increased oxidative metabolism resulting from the need for compensation of altered inputs from gravity-sensitive epithelia in the inner ear. Another possibility is that acceleration within a centrifuge causes physiological stress for the animals and, therefore, influences the cytochrome oxidase activity in neurons.

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.

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.

Calcium localization in the shell-forming tissue of the freshwater snail, Biomphalaria glabrata: a comparative study of various methods for localizing calcium.

The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snail Biomphalaria glabrata (Planorbidae, Basommatophora). In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue. The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.

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.

Synaptic plasticity and gravity: ultrastructural, biochemical and physico-chemical fundamentals.

On the basis of quantitative disturbances of the swimming behaviour of aquatic vertebrates ("loop-swimming" in fish and frog larvae) following long-term hyper-g-exposure the question was raised whether or not and to what extent changes in the gravitational vector might influence the CNS at the cellular level. Therefore, by means of histological, histochemical and biochemical analyses the effect of 2-4 x g for 9 days on the gross morphology of the fish brain, and on different neuronal enzymes was investigated. In order to enable a more precise analysis in future-microgravity-experiments of any gravity-related effects on the neuronal synapses within the gravity-perceptive integration centers differentiated electron-microscopical and electronspectroscopical techniques have been developed to accomplish an ultrastructural localization of calcium, a high-affinity Ca2(+)-ATPase, creatine kinase and cytochrome oxidase. In hyper-g animals vs. 1-g controls, a reduction of total brain volume (15%), a decrease in creatine kinase activity (20%), a local increase in cytochrome oxidase activity, but no differences in Ca2+/Mg(2+)-ATPase activities were observed. Ultrastructural peculiarities of synaptic contact formation in gravity-related integration centers (Nucleus magnocellularis) were found. These results are discussed on the basis of a direct effect of hyper-gravity not only on the gravity-sensitive neuronal integration centers but possibly also on the physico-chemical properties of the lipid bilayer of neuronal membranes in general.

The application of energy-filtering electron microscopy for the cytochemical localization of Ca(2+)-ATPase activity in synaptic terminals.

The energy-filtering electron microscopical modes of electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) have been applied to the cytochemical detection of Ca(2+)-ATPase activity in synaptic terminals in the brain of a cichlid fish. Using a recently developed modification of an enzyme-histochemical method, cerium phosphate was precipitated as a marker of high-affinity Ca(2+)-ATPase activity. This is considered to be a marker for the plasmalemma-bound calcium pump, an enzyme which plays a crucial role in the regulation of the cytoplasmic calcium concentrations and therefore of the reactivity of nerve cells. High-affinity Ca(2+)-ATPase activity is located preferentially at the inner side of synaptic plasma membranes and enables a discrimination of different types of synapse. It is only by using EELS and ESI that the very small amounts of high-affinity Ca(2+)-ATPase reaction product can be analysed reliably and located precisely. These new electron microscopical techniques offer powerful tools for cytochemical studies.

Morphogenetic development of the area octavolateralis in the cichlid fish Oreochromis mossambicus.

In the cichlid fish Oreochromis mossambicus the area octavolateralis in the brain stem was studied using histological techniques and labelling of afferents from the inner ear and the lateral line system with horseradish peroxidase (HRP). Adult fish and fish larvae aged 2, 4, 7, 10, and 15 days post hatching were investigated in order to analyze the ontogenetic development of these sensory systems during the critical phase of maturation from relatively immobile stages to actively swimming larvae. In adult Oreochromis seven nuclei can be detected in the octavolateral area while in the larvae a steady differentiation from a mostly periventricular concentration of perikarya in the first two days post hatching via increasing accumulations of neuronal cell bodies in the more peripheral regions up to four discernible nuclei in the age of 15 days post hatching was observed. The HRP-labelling of afferents from the lateral line organ and the inner ear in early stages indicates that the sensory organs precedes the histologically detectable differentiation in the octavolateral area. Both sensory components are clearly separated already in very early developmental stages.