Life Sciences Commons^™

The Role Of Calcium In The Regulation Of Apoptosis, David J. Mcconkey

Scanning Microscopy

The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegans to humans suggests that a conserved set of biochemical mechanism(s) may also be involved in the response. Early evidence suggested that the endogenous endonuclease implicated in apoptosis in most model systems is Ca2+ -dependent, and subsequent work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis are one of the conserved biochemical pathways regulating the response. Molecular targets for Ca2+ are now being identified and include signal transduction intermediates, endonuclease(s) and proteases, and the enzymes involved in the …

Applications Of Laser Microprobe Mass Spectrometry In Biology And Medicine, Annette Bakker, Luc Van Vaeck, Wim Jacob

Scanning Microscopy

Laser microprobe mass spectrometry (LMMS) provides spot analysis with a lateral resolution of 1-5 μm. Focused laser ionisation, combined with mass spectrometry, yields information on elements, molecular identification of inorganic substances, and structural characterisation of organic molecules of typically less than 2-3 kDa. Quantification in complex heterogeneous systems, such as biological materials, is not possible at the present state of the art. The strength of the method lies in the qualitative information on the molecular composition of the analyte, not just element detection, with a lateral resolution at the light microscopy level. The applications of this technique in the field …

Impact Of Escherichia Coli On Urine Citrate And Urease-Induced Crystallization, A. Edin-Liljegren, H. H. Hedelin, L. Grenabo, S. Pettersson

Scanning Microscopy

Escherichia coli (E. coli) is usually not a urease producer. It is, however, often cultured in urinary phosphate containing calculi including ammonium magnesium phosphate stones. This suggests the possibility that E. coli might be involved in stone forming process. The effect of E. coli on urine citrate and urease-induced crystallization in human urine has been studied in vitro. E. coli was found to strongly reduce urine citrate (after 48 hours). In the E. coli inoculated samples, the urease-induced crystallization was increased. There was a strong correlation, r = 0.8, between the citrate decrease and the increase in calcium precipitation. The …

Confocal Fluorescence Ratio Imaging Of Ion Activities In Plant Cells, M. D. Fricker, M. Tlalka, J. Ermantraut, G. Obermeyer, M. Dewey, S. Gurr, J. Patrick, N. S. White

Scanning Microscopy

Fluorescent probes allow measurement of dynamic changes of calcium and pH in living cells. Imaging using confocal scanning laser microscopy provides a route to spatially map these dynamics over time in single optical sections or in 3-D images. We have developed a dual-excitation confocal system to allow ratio measurements of pH and calcium, that compensate for changes in dye distribution, leakage and photobleaching. Application of these techniques to plant tissues is complicated by the difficulty in loading the tissues with dye. We describe a new technique to assist dye loading in intact leaves of Lemna using a pre-treatment with cutinase. …

Proton-Induced And Electron-Induced X-Ray Microanalysis Of Insulin-Secreting Cells, Eva Pålsgård, Ulf Lindh, Lisa Juntti-Berggren, Per-Olof Berggren, Godfried M. Roomans, Geoffrey W. Grime

Scanning Microscopy

Elemental redistribution induced by insulin secretion, was investigated by electron and proton probe X-ray microanalysis. In particular, ion fluxes following immediately upon stimulation were studied. As the sensitivity of the electron probe was insufficient, the proton microprobe was employed. In order to see whether the cell is asymmetric with respect to Ca²⁺ influx, the cells were stimulated in the presence of Sr²⁺ (as a Ca²⁺ analog). Insulin-secreting cells (RINm5F cells and isolated mouse β-cells) were cultured on grids and shock-frozen at 2-30 seconds after stimulation. In a large number of cells, the major elements and and large …

Standards For X-Ray Microanalysis Of Calcified Structures, J. A. Lopez-Escamez, A. Campos

Scanning Microscopy

The ability of electron probe X-ray microanalysis (EPMA) to solve biological problems often depends on the use of a quantitative approach. EPMA allows the quantitative determination of chemical elements of biological materials by using reference standards which resemble the specimen in the mode of interaction with the electron beam. Although there is a large experience in the quantification of elements in biological thin specimens, experience with standards for X-ray microanalysis of bulk specimens is limited, especially for calcified structures where the density of the specimen is difficult to estimate. The quality of the results in EPMA depends on obtaining accurate …

Biological Electron Energy Loss Spectroscopy In The Field-Emission Scanning Transmission Electron Microscope, R. D. Leapman, S. Q. Sun, J. A. Hunt, S. B. Andrews

Scanning Microscopy

The dedicated scanning transmission electron microscope (STEM) combined with parallel electron energy loss spectroscopy (EELS) provides a very sensitive means of detecting specific elements in small structures. EELS is more sensitive than optimized energy-dispersive X-ray spectroscopy by a factor of about three for calcium. Measurement of such low concentrations requires special processing methods such as difference-acquisition techniques and multiple least squares procedures for fitting reference spectra. By analyzing data recorded at each pixel in a spectrum-image it is possible to map quantitatively the elemental distributions in a specimen. It is possible to prepare cryosections that are sufficiently thin to avoid …

Image-Eels: A Synthesis Of Energy-Loss Analysis And Imaging, Karl-Heinz Körtje

Scanning Microscopy

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 …

Optical Methods For Imaging Ionic Activities, Roger B. Moreton

Scanning Microscopy

Optical fluorescence is characteristic of molecules and their environment, and dyes can be made whose fluorescence is altered by reversible binding to specific ions. By introducing these into the cytosol, fluorescence microscopy can be used to form dynamic images of ionic activities in living cells under experimental manipulation. Optical fluorescence spectra are broad-band, and if specific ion binding alters the wavelength of maximal excitation or emission, quantitative measurements can be made from the ratio of images taken at two different wavelengths, eliminating errors due to spatial variations in dye concentration and optical path-length. This method is analogous to continuum normalisation …

Laser Microprobe Mass Spectrometry In Biology And Biomedicine, S. Eeckhaoudt, L. Van Vaeck, R. Gijbels, R. E. Van Grieken

Scanning Microscopy

An overview is given of laser microprobe mass spectrometry (LMMS) in biology and biomedicine (1989-1993). The present instrumentation and its analytical features are surveyed. Applications are presented with special attention on human and animal tissue samples, as well as plant material. The capabilities of LMMS to study the element distribution in histological sections, to identify the chemical composition of inorganic inclusions and to generate structural information from organic compounds are evidenced.

Measurement Of Subcellular Ca2+ Redistribution In Cardiac Muscle In Situ: Time Resolved Rapid Freezing And Electron Probe Microanalysis, Meredith Bond, Mark D. Schluchter, Eva Keller, Christine S. Moravec

Scanning Microscopy

To directly assess the physiological roles of sarcoplasmic reticulum (SR) and miitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca²⁺ during the cardiac contractile cycle. Changes in Ca²⁺ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca²⁺ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of …

X-Ray Microanalysis Of Chief Cells In Rat Parathyroid Gland, Romuald Wroblewski

Scanning Microscopy

X-ray microanalysis was used to study the elemental composition of the rat parathyroid gland. Analyses were performed in morphologically defined cells. Use of semi-thin cryosections allowed us to selectively analyze cell nucleus, cytoplasm and occasionally dense granules in the cytoplasm.

The concentration of elements (mmol/kg dry weight) in chief cells of parathyroid gland were as follows: Na 83, Mg 48, P 853, S 224, Cl 164, K 551 and Ca 10. Significantly higher phosphorus and potassium concentrations were found in the dense granules as compared to cytoplasm of the parathyroid chief cells. Choosing rat parathyroid gland gave the possibility to …

Analysis Of Early Hard Tissue Formation In Dentine By Energy Dispersive X-Ray Microanalysis And Energy-Filtering Transmission Electron Microscopy, Hans-Peter Wiesmann, Ulrich Plate, Hans-Jürgen Höhling, Rudolf H. Barckhaus, Karl Zierold

Scanning 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 …

Strontium, A Tracer To Study The Transport Of Calcium In Mineralizing Tissues By Electron Probe Microanalysis, Ernst-Rudolf Krefting, Kai Frentzel, Jörg Teßarek, Hans-Jürgen Höhling

Scanning Microscopy

In growth plate cartilage the mineralization starts extracellularly in the lower hypertrophic zone. The mineral formed is the calcium phosphate apatite. Enough calcium and phosphate must be available at the mineralization front as well as in regions with proceeding mineralization. There must be a transport of Ca (and phosphate) to these sites.

Electron probe X-ray microanalysis is a well established method to analyze element concentrations in small volumes, but it cannot discriminate isotopes. Strontium is similar to Ca in its chemical and biological behaviour and is therefore a suitable tracer to investigate the transport of Ca.

Small amounts of Sr …

Electron Probe Microanalysis Of The Otolithic Membrane. A Methodological And Quantitative Study, J. A. López-Escámez, F. J. Cañizares, P. V. Crespo, A. Campos

Scanning Microscopy

The effect of tissue preparation on calcium and potassium weight percents in the otoconial layer in the utricle and saccule was studied in four groups of OF1 mice with electron probe X-ray microanalysis. Glutaraldehyde and freeze-drying, glutaraldehyde and air-drying, air-drying, and cryo-fixation and freeze-drying were compared. Ca and K changed significantly in the utricle depending on the method used (P < 0.001), and K changed significantly in the saccule (P < 0.001 ). We chose cryo-fixation with freeze-drying for the quantitative analysis of the otolithic membrane because this method provided the highest values of Ca and K with minimum loss of Ca and K . Microcrystalline salt standards mounted on scanning electron microscopy holders were used for the quantification of Ca and K by the peak-to-local-background (P/B) ratio method. The P/B ratio in standards with reproducible results, when plotted against weight percent, gave a straight line for Ca (r = 0.99, P < 0.001) and K (r = 0.98, P < 0.001). The Ca and K weight percents in otoconia showed similar frequency distributions in the utricle and saccule.

Histochemical Demonstration And Microanalysis Of Possible Calcium Binding Sites In The Enamel Organ Of Rat Incisors, Y. Takano

Scanning Microscopy

The rat incisors obtained from rats perfused with high-calcium solution containing 30 to 50 mM CaCl₂ were processed for rapid freeze/freeze-substitution and embedded in epoxy resin. GBHA staining, a histochemical staining for calcium, of unhydrously prepared sections revealed a large number of granular Ca-GBHA reactions in the enamel organ, most of which being located along the lateral plasma membranes of the ameloblasts. In the ameloblast layer, the reaction was negative in the presecretory stage, became intense in concert with the onset of enamel matrix formation, and remained so by the end of the transitional stage where the reaction gradually …

The Influence Of Dietary Factors On The Risk Of Urinary Stone Formation, A. Hesse, R. Siener, H. Heynck, A. Jahnen

Scanning Microscopy

The action of various beverages and foods on the composition of the urine in the circadian rhythm and in the 24-hour urine has been investigated under standardized conditions. Orange juice leads to a significant increase of urinary pH and citric acid excretion. Black tea leads to a raised excretion of oxalic acid by only 7.9%. In the short term, beer increases diuresis, but afterwards leads to a compensatory antidiuresis with increased risk of stone formation. Depending on their composition, mineral waters have very different effects on the urinary constituents. Milk as well as cocoa beverage significantly increase calcium excretion; moreover, …

Particle Induced X-Ray Emission Microanalysis Of Root Samples From Beech (Fagus Sylvatica), M. Hult, B. Bengtsson, N. P. -O. Larsson, C. Yang

Scanning Microscopy

Beech seedlings (Fagus Sylvatica) were grown in a nutrient solution to which AlCl₃ had been added. The experiments were designed to evaluate the effect of aluminum on the localization of elements in plant root tissue. Cross-sections of roots were analyzed with micro-PIXE (particle induced X-ray emission) and Rutherford backscattering (RBS) simultaneously. A proton beam of either 30x30 μm² or 5x5 μm² was scanned over the samples, and quantitative elemental maps were created. Aluminum was found to accumulate in high concentrations (percent levels) in the epidermis and the outer layer of the cortex, when the nutrient …

Factors Affecting Fasting Urinary Calcium Excretion In Stone Former Patients On Different Dietary Calcium Intake, Piergiorgio Messa, Giuseppe Mioni, Rossana Franzon, Michele Messa, Aldo Cruciatti, Sandro Giannini, Angela D'Angelo

Scanning Microscopy

The effects of variable calcium content on daily and fasting urinary calcium and other lithogenic solutes excretion, on the bone turnover index (fasting hydroxyproline urinary excretion) and on the calciotropic hormones were studied in 312 stone former patients with an outpatient protocol and 15 stone former patients in an inpatient study. Furthermore in 60 of these patients, 30 while on a low calcium diet (LCD) and 30 on a free calcium home diet (FCD), the effects of an oral calcium load (OCL) on bone turnover index, calciotropic hormones and calcium excretion were evaluated. The results demonstrate that an LCD is …

Changes In Sodium Concentration In Cardiac Myocytes From Diabetic Rats, Alice Warley

Scanning Microscopy

The effects of streptozotocin induced diabetes on rats were studied. The animals showed an increase in blood glucose concentration and a loss of weight from both the body and the heart. Loss of weight from the heart was less severe leading to an increased heart to body weight ratio. Study of element concentrations by X-ray microanalysis showed that there was an increase in intracellular Na concentration in cardiac myocytes from the diabetic animals, but no change in Mg. These results agree with studies which show changes in Na/K ATPase after the onset of diabetes.

Microprobe Analyses Of The Potassium-Calcium Distribution Relationship In Predentine, H. J. Höhling, H. Mishima, Y. Kozawa, T. Daimon, R. H. Barckhaus, K. -D. Richter

Scanning Microscopy

Apex regions of continuously growing incisors of Wistar rats were quickly dissected, shock-frozen in liquid nitrogen-cooled propane, freeze-dried at -80 °C and infiltrated with Spurr's resin . 400nm thick dry sections were cut with a diamond knife on an ultramicrotome . Relatively flat sections were transferred with an eye lash onto collodium coated aluminum grids. They were flattened with a glass stick and by placing another collodi um coated aluminum grid just on top of the first one, exerting a uniform pressure . After carbon coating the sections were observed using the backscattered and secondary electron signals in a scanning …

Micro-Pixe (Proton-Induced X-Ray Emission) Study Of The Effects Of Fluoride On Mineral Distribution Patterns In Enamel And Dentin In The Developing Hamster Tooth Germ, D. M. Lyaruu, G. H. J. Tros, A. L. J. J. Bronckers, J. H. M. Wöltgens

Scanning Microscopy

Micro-PIXE (proton-induced X-ray emission) analysis was performed on unfixed and anhydrously prepared sections from developing enamel and dentin from hamsters injected with a single dose of 20 mg NaF /kg body weight. Fluoride, apart from inducing the formation of the characteristic paired response in the enamel (i.e., a hyper- followed by a hypomineralized band in the secretory enamel), also induces the formation of sub-ameloblastic cystic lesions under the transitional and early secretory enamel accompanied by relatively intense hypermineralization of the underlying cystic enamel surface. These cystic lesions, however, were only found to be associated with certain isolated populations of these …

X-Ray Microanalysis Of Ca And K In Corn Bran And Oat Hulls, F. R. Dintzis, F. L. Baker, T. C. Nelsen

Scanning Microscopy

Oat hulls and dry-milled corn bran were loaded with calcium or potassium and made into either sectioned bulk specimens of intact tissue embedded in resin or into non-sectioned bulk specimens made from powdered-compressed tissue formed into disks without resin. Regression lines of X-ray count versus mineral concentration were similar for both Ca and K. X-ray count versus mineral concentration relationships were similar for intact oat hulls and powdered-compressed specimens of either oat hulls or corn bran. However, the relationship for intact corn bran embedded in resin was significantly different. While the reason for this difference is not known, the result …

Techniques For The Microanalysis Of Higher Plants With Particular Reference To Silicon In Cryofixed Wheat Tissues, M. J. Hodson, A. G. Sangster

Scanning Microscopy

The applications of x-ray microanalysis in research into silicon in higher plants are reviewed, recent developments are assessed, and new data are presented. Conventionally prepared material [air or freeze drying for scanning electron microscopy (SEM), and glutaraldehyde/osmium tetroxide fixation for transmission electron microscopy (TEM)) has been studied using both wavelength and energy dispersive microanalysis. These techniques are reliable provided that the deposited form of silica is the major focus of investigation. Recently, studies concerning the soluble, mobile forms of silica, and the ionic environment at deposition sites have been initiated. In these investigations x -ray microanalysis has been carried out …

Strontium And Bromide As Tracers In X-Ray Microanalysis Of Biological Tissue, Joanna Wroblewski, Samuel Sagström, Helmy Mulders, Godfried M. Roomans

Scanning Microscopy

Since energy dispersive X-ray microanalysis cannot distinguish between isotopes of the same element, alternative methods have to be used to get information similar to that obtained in experiments with radioactive tracers. In the present study, strontium was used as a tracer for calcium, and bromide as a tracer for chloride. Rats were injected with strontium chloride in vivo, and the uptake of strontium in the acinar cells of the submandibular gland was studied. Eventually a Sr/Ca ratio of 0.3 was reached. In some animals, secretion of mucus had been elicited by stimulation with isoproterenol 4 h prior to injection …

Bone Lining Cells: Structure And Function, Scott C. Miller, Louis De Saint-Georges, Beth M. Bowman, Webster S. S. Jee

Scanning Microscopy

Bone lining cells (BLC's) cover inactive (nonremodeling) bone surfaces, particularly evident in the adult skeleton. BLC's are thinly extended over bone surfaces, have flat or slightly ovoid nuclei, connect to other BLC's via gap junctions, and send cell processes into surface canaliculi. BLC's can be induced to proliferate and differentiate into osteogenic cells and may represent a source of "determined" osteogenic precursors. BLC's and other cells of the endosteal tissues may be an integral part of the marrow stromal system and have important functions in hematopoiesis, perhaps by controlling the inductive microenvironment. Because activation of bone remodeling occurs on inactive …

Application Of The Pyroantimonate Method And Electron Probe Microanalysis To The Study Of Glycogen Metabolism In Liver, Pascale Mentré, Sylvain Halpern

Scanning Microscopy

Glycogen distribution in the liver of mouse under different metabolic conditions was studied by the pyroantimonate (PA) method combined with semi-quantitative electron probe microanalysis (EPMA). In the liver of animals subjected to a sugar-rich diet, glycogen granules were abundant and electron transparent. In fasted animals, they were less numerous and stained by PA, which indicates the presence of a complexed cation. This cation was identified as calcium by EPMA. In both cases, adjacent cytoplasmic areas contained "masked" calcium not revealed by PA but detected by EPMA, which is characteristic of a neutral complexed form; but in the case of the …

Calcium Levels In Ruffle-Ended And Smooth-Ended Maturation Ameloblasts, S. H. Ashrafi, D. R. Eisenmann, A. E. Zaki

Scanning Microscopy

Scanning electron microscopy was used to distinguish the topographical characteristics of two maturation ameloblast types in freeze-dried blocks of enamel organ tissue. This distinction was based primarily upon the configuration of the distal ends of the ameloblasts and the presence or absence of wide intercellular spaces.

Energy dispersive x-ray spectrometry was applied to compare calcium levels in various regions of tissue identified as constituting either ruffle-ended or smooth ended ameloblasts. Greater levels of calcium were found in the distal ends of the ruffle-ended cells than in their proximal ends. In addition, greater calcium levels were found in the distal ends …

X-Ray Microanalysis Of Calcium Containing Organelles In Resin Embedded Tissue, G. Nicaise, I. Gillot, A. K. Julliard, E. Keicher, S. Blaineau, J. Amsellem, J. C. Meyran, M. L. Hernandez-Nicaise, B. Ciapa, C. Gleyzal

Scanning Microscopy

The localization of calcium in cell organelles at the electron microscope level is often achieved through cytochemical techniques, and verified by X-ray microanalysis. Various methods have been used to cytochemically detect calcium or calcium-binding sites : calcium loading, calcium substitution by strontium, barium, or even lead, and calcium precipitation by oxalate, phosphate, fluoride, or pyroantimonate. Their results may have heuristic value, particularly in preliminary studies of poorly known cell types. A complementary and more physiological approach is offered by quantitative measurement of the total calcium content of organelles after cryofixation.

Resin embedding is less demanding than cryomicrotomy and gives better …

X-Ray Analysis Of Biological Fluids: An Update, G. A. Quamme

Scanning Microscopy

X-ray analysis has been used for some twenty years to quantify elements including Na, K, Cl, Ca, Mg and phosphorous in biological fluid samples. The method consists of accurate pipetting of 50-150 pl samples and appropriate drying to form crystalline deposits. Quantitation is obtained by comparison of x-ray intensities with comparable standard crystalline deposits. Electon microprobe analyses have contributed much to our understanding of renal transport function including physiological controls and pathological influences on the tubular handling of the major elements. New approaches to x-ray analysis of solid tissue will further our knowledge concerning transport properties in the kidney and …