Biology Commons^™

Ultrastructural Observations Of The Argonaut Shell, P. R. Mitchell, P. P. Phakey, W. A. Rachinger

Scanning Microscopy

An examination of the ultrastructure of the shell of the cephalopod Argonauta Nodosa was carried out using scanning electron microscopy, transmission electron microscopy and polarised light microscopy. The structure of the Argonaut shell was found to consist of an inner and outer prismatic layer separated by a thin central zone which was sparsely occupied by spherulitic crystals. Fluctuations in the width and porosity of the central zone resulted in changes in the shell's opacity and gave rise to the fibrous lines visible in the structure. The central zone was the region of initial growth and was the nucleating point for …

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 …

Electron Probe X-Ray Microanalysis Of Epithelial Cells: Aspects Of Cryofixation, Karl Zierold, Hartmut Hentschel, Frank Wehner, Armin Wessing

Scanning Microscopy

Content and distribution of diffusible ions in epithelial cells were studied by scanning transmission electron microscopy and energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections from trout kidney, rat liver and Malpighian tubules of Drosophila larvae. Cryofixation of small excised kidney and liver samples by rapid immersion into liquid propane resulted in intracellular K/Na-ratios < 1. In contrast, K/Na-ratios > 7 were obtained after in situ cryofixation by means of a cryopunching device which allows tissue pieces to be frozen during excision from the intact organ. Isolated hepatocytes cryofixed in a small droplet of culture medium had a K/Na-ratio of 3.7. After culturing the hepatocytes, …

Radiation-Induced Fibrosis: The Structure/Function Relationship, J. C. Murray

Scanning Microscopy

Fibrosis is a frequent complication of therapeutic radiation delivered to organ sites such as the thorax and pelvic region. We investigated the relationship between function, and biochemical and structural changes in the lung and colon of CBA mice irradiated with 240 kV X-rays. Progressive changes in lung function, as evidenced by increased breathing rates were observed out to 6 months post-irradiation. Biochemical studies demonstrated increased metabolism (synthesis and breakdown) of collagen around 6 months after irradiation, but provided no evidence of net collagen accumulation. Analysis of collagen isotypes revealed a slight increase in the ratio of types I and Ill. …

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 …

Crystallization Of Calcium Oxalate And Calcium Phosphate At Supersaturation Levels Corresponding To Those In Different Parts Of The Nephron, Ján Lupták, Hanne Bek-Jensen, Anne-Marie Fornander, Inge Höjgaard, Mari-Anne Nilsson, Hans-Göran Tiselius

Scanning Microscopy

The risk of crystallization in solutions, with a composition corresponding to that of urine in various parts of the nephron, was assessed by studying urine samples containing standardized increments of calcium and oxalate concentrations. The experiments were carried out in salt solutions with and without dialysed urine and the appearance of crystals was detected and measured with a Coulter counter. With increased concentrations of calcium, crystals of calcium phosphate were predominantly formed in solutions with a composition corresponding to that in the proximal and distal tubuli, whereas, calcium oxalate was the primary crystallization product in solutions with a composition corresponding …

Effects Of Acute Acid Loading On The Risk Of Calcium Phosphate And Calcium Oxalate Crystallization In Urine, P. J. Osther, J. Bollerslev, J. R. Nørgård, K. Engel, P. Kildeberg

Scanning Microscopy

The aim of this study was to examine the risk of calcium phosphate and calcium oxalate crystallization during acute acid loading under controlled conditions.

The effects of acute acid loading on rates of renal excretion of calcium, magnesium, phosphate, citrate, oxalate and urine pH were studied in healthy subjects. The risk of calcium phosphate and calcium oxalate crystallization were evaluated by estimates of the ion activity products of calcium phosphate [AP(CaP)-index] and calcium oxalate [AP(CaOx)-index] according to Tiselius. In addition, the risk of brushite [AP(Bru)-index] crystallization was estimated.

An acute acid load administered as ammonium chloride (NH₄Cl) produced …

Changes Of Ion And Water Content Of Mouse Intestinal Cells After Pilocarpine And Isoproterenol Stimulation, Thomas Von Zglinicki, Godfried M. Roomans

Scanning Microscopy

Cytoplasmic monovalent ion and water contents in morphologically defined mice jejuna) cells were measured by X-ray microanalysis in order to gain insight into the cell-type specificity of intestinal electrolyte transport mechanisms. Ion and water contents were measured independently. It was found that in some cases net fluxes of ions and water do not correspond to the assumption of constant osmotic activity of cytoplasmic Na and K ions. Stimulation of secretion with the cholinergic secretagogue pilocarpine resulted in efflux of Cl^- from and influx of K⁺ into crypt enterocytes containing small secretion granula (crypt A cells). No significant changes …

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.

X-Ray Microanalysis With The Environmental Scanning Electron Microscope: Interpretation Of Data Obtained Under Different Atmospheric Conditions, D. C. Sigee, C. Gilpin

Scanning Microscopy

X-ray microanalysis of non-biological and biological specimens was carried out in an environmental scanning electron microscope (ESEM) over a range of atmospheric conditions. Introduction of water vapour into the specimen chamber lead to direct X-ray contribution from oxygen atoms, an increase in extraneous background (causing reduced P/B ratios of other elements), X-ray absorption (also reducing P/B ratios) and broadening (skirting) of the electron beam. Similar results were obtained after introduction of an argon atmosphere. These effects were reduced under conditions of minimal chamber atmospheric pressure and maximal accelerating voltage.

Because of beam skirting, quantitative X-ray microanalysis of biological specimens in …

Ion Microscopy In Biology, George H. Morrison, Isabelle Gay, Subhash Chandra

Scanning Microscopy

Ion microscopy, a mass spectrometry based isotopic imaging technique, is uniquely suited for ion transport-related problems in biological systems. Due to its high sensitivity, it can image the transport and distribution of both major and minor elements (isotopes) at subcellular resolutions. The images of major elements such as K, Na, Cl, etc., can be viewed directly and recorded in real-time from the microchannel plate-fluorescent screen detector of the instrument. The low concentration physiologically important elements, such as Ca, need about one minute of integration for good quality imaging. The isotopic imaging capability of ion microscopy provides a unique approach for …

Use Of In Vitro Systems For X-Ray Microanalysis, Jarin Hongpaisan, Ann-Christin Mörk, Godfried M. Roomans

Scanning Microscopy

The use of X-ray microanalysis in human pathology may require the use of cryoprepared tissue. Often it is impossible to carry out freezing of the tissue in an optimal way, and in addition, it is difficult to carry out experiments in living patients. The use of in vitro systems and cell cultures allows separation of the process of tissue removal and the freezing procedure, and also makes testing of pharmacological or toxic substances possible. In experiments with animal tissue it was shown that incubation in a physiological buffer induced significant changes in the concentrations of Na, K, and Cl. In …

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 …

Standardless Analysis Of Biological Tissue Sections, W. A. P. Nicholson

Scanning Microscopy

The X-ray microanalysis of thin biological samples which are usually supported on a thin organic film or are self-supporting specimens, has required the use of standards which contain the elements of interest. Spectra from the standards are used to calculate the factors for converting X-ray data recorded on the specimen into elemental concentrations. A method is discussed here, in which these factors are evaluated from formulae. The most important physical process to be evaluated is that of characteristic X-ray production in the specimen. The bremsstrahlung production must also be evaluated if the Hall or continuum normalisation (CN) method of quantitation …

Low Temperature Embedding Of Chemically Unfixed Biological Material After Cryosorption Freeze-Drying, L. Edelmann

Scanning Microscopy

After freeze-drying of bulk specimens in a newly developed cryosorption freeze-dryer (CFD) a special accessory is used to infiltrate the specimens in Lowicryl HM20 and to polymerize them at low temperature by ultra-violet (UV) irradiation within the CFD chamber in flat embedding moulds. The accessory allows polymerization in a dry, oxygen free environment without the risk of evaporation of volatile components of the resin which may lead to unsatisfactory polymerization. First results demonstrate the quality of structure preservation of biological material not treated with any chemical fixative.

Effects Of Ionizing Radiation On The Size Distribution Of Proteoglycan Aggregates Synthesized By Chondrocytes In Agarose, M. Cornelissen, H. Thierens, L. De Ridder

Scanning Microscopy

Effects of ionizing radiation on the structure and size-distribution of newly synthesized proteoglycan aggregates are studied in-vitro. Chondrocytes, isolated from embryonic chick sterna, are cultured for 7 days in a tri-dimensional agarose system. Single radiation doses of 10, 20 and 50 Gy are given before starting the culture. Digestion of the artificial agarose matrix liberates the newly synthesized proteoglycans. Spreading with cytochrome C allows electron microscopic investigations of the individual, newly synthesized molecules.

The structure of aggregates synthesized by control and irradiated chondrocytes is comparable. However, radiation causes alterations in the size-distributions of the aggregate-populations. For the control cultures, an …

Investigation Of Dislocations In Gaas Using Cathodoluminescence In The Scanning Electron Microscope, K. L. Pey, J. C. H. Phang, D. S. H. Chan

Scanning Microscopy

Electrically active dislocations in Si-doped {100} GaAs substrates were observed using the cathodoluminescence (CL) technique in the scanning electron microscope (SEM). CL contrast profiles were experimentally obtained from the dislocations at different beam energies. Based on the CL model for localized defects in semiconductors developed earlier by Pey, the depths of the dislocations were found by locating the beam energy at which maximum CL contrast occurred. A preferential etching technique for {100} GaAs was employed to reveal the dislocations and to measure their depths. The etched depths obtained were compared to the predicted results from the theoretical model developed. The …

Imaging-Sims (Secondary Ion Mass Spectroscopy) Studies Of Advanced Materials, R. Levi-Setti, J. M. Chabala, J. Li, K. L. Gavrilov, R. Mogilevsky, K. K. Soni

Scanning Microscopy

This article describes the application of scanning ion microprobe (SIM) microanalysis for the characterization of advanced engineered materials. In conjunction with secondary ion mass spectrometry (SIMS), scanning ion microprobes can image elemental distributions over surfaces with high lateral resolution (50-100 nm). With this technique, most elements, including isotopes, can be detected with good sensitivity. The principles and instrumentation associated with the SIM/SIMS technique are briefly described and ongoing developments are outlined. The analytical capabilities of the technique are illustrated by case studies of aluminum-lithium alloys, zinc oxide varistors, aluminum matrix composites, and photographic materials.

Quantitative Elemental Mapping Of Biomedical Specimens Using The Nuclear Microprobe., Jan Pallon, John Knox

Scanning Microscopy

Quantitative and structural elemental information is available from the nuclear microprobe through a time consuming fit of the (X-ray) spectrum at each point. An alternative technique is proposed which greatly shortens the analysis procedure and allows an increased number of samples to be processed. The method discussed here is to create elemental maps which, when they are divided by the charge/pixel and multiplied by a scaling factor, will form quantitative maps. The scaling factors are obtained from a calibration procedure comparing a large number of fitted X-ray spectra with the corresponding contents of selected energy windows. The technique also allows …

Ischemia/Reperfusion Injury Of The Ascending Colon In Ponies: A Correlative Study Utilizing Microvascular Histopathology And Corrosion Casting, B. J. Darien, P. A. Sims, W. C. Stone, D. R. Schilly, R. R. Dubielzig, R. M. Albrecht

Scanning Microscopy

Volvulus of the ascending colon (ACV) in the horse results in microvascular injury and necrosis of the intestinal mucosa. This study investigated the site and type of microvascular injury which occurs within the mucosa and sub mucosa following ACV. Histopathology of volvulus treated ponies demonstrated mucosa! necrosis with microvascular hemorrhage and thrombosis. Thrombi occurred within the subepithelial capillaries and edema and hemorrhage developed throughout the mucosa and submucosa. Vascular casts allowed 3-D viewing of samples obtained from the entire pelvic flexure and demonstrated two distinct microvascular changes: 1) disruption of the colonic glandular capillary network occurred concomitantly with the …

Improved Visualization Of Dna In Aqueous Buffer With The Atomic Force Microscope, Magdalena Bezanilla, Carlos J. Bustamante, Helen G. Hansma

Scanning Microscopy

An improved method has been developed for imaging deoxyribonucleic acid (DNA) in aqueous buffer with the atomic force microscope (AFM). DNA on untreated mica can be imaged in aqueous buffer with the AFM if the DNA is deposited onto the mica in a buffer with HEPES and MgCl₂, if the sample is rinsed thoroughly with high water pressure, and if the imaging is done with an electron beam-deposited (EBD) tip that has been deposited in the scanning electron microscope (SEM). The water rinse removes DNA that is otherwise easily scraped off the substrate. There is evidence that sharper …

Caesium On Si(100) Studied By Biassed Secondary Electron Microscopy, M. Azim, C. J. Harland, T. J. Martin, R. H. Milne, J. A. Venables

Scanning Microscopy

An ultra-high vacuum scanning electron microscope (UHV-SEM) has been used to study sub-monolayers of Cs on Si(100) surface. Cs adsorption on the surface causes a considerable change in the work function. Coverages below 1/2 monolayer (ML) have been estimated by correlating the work function changes with the secondary electron (SE) signal. It has been found that this signal is sensitive down to ~ 0.005 ML when the sample is biassed to a few hundred volts.

Electron trajectories from a biassed sample have been simulated for electrons originating from different areas with different work functions across the sample. This indicates that …

Effects Of Modulated And Continuous Microwave Irradiation On Pyroantimonate Precipitable Calcium Content In Junctional Complex Of Mouse Small Intestine, Z. Somosy, G. Thuróczy, J. Kovács

Scanning Microscopy

The pyroantimonate precipitable calcium content of intestinal epithelial cells was investigated in mice following total body irradiation with 2450 MHz continuous and low frequency (16 Hz) square modulated waves. In the control animals the reaction products appeared in the intercellular space of adjacent cells including intermediate junctions and desmosomes and were absent in the area of tight junctions. Immediately after low frequency modulated microwave irradiation at 0.5 and 1mW/cm² power densities, a rapid distribution of pyroantimonate precipitable calcium content was observed. The pyroantimonate deposits were located on the cytoplasmic side of lateral membrane, in the area of junctional complex, …

Calcium Phosphate In Aspergillosis Of The Maxillary Sinus, Hidekuni Tanaka, Toshiro Sakae, Hiroyuki Mishima, Hirotsugu Yamamoto

Scanning Microscopy

The unique appearance of apatite in fungus balls of aspergillosis in the maxillary sinus was investigated using scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), X-ray microdiffraction and Fourier transform infrared spectroscopy (FT-IR). Rod-shaped fragments with tubular structures, and globular vesicles covered with conidia were observed in the fungus balls. Massive fragments of a solid substance were demonstrated inside the fungus balls. Calcium and phosphate were detected in necrotic areas of the fungus balls by EDS. X-ray microdiffraction and FT-IR showed the presence of an apatite-like substance, but failed to demonstrate the presence of calcium oxalate crystals usually found in such …

X-Ray Microanalysis Of Camp-Induced Ion Transport In Ncl-Sg3 Sweat Gland Cells, Ann-Christin Mörk, Godfried M. Roomans

Scanning Microscopy

cyclic AMP (cAMP)-induced ion transport in a human sweat gland cell line, NCL-SG3, was investigated by X-ray microanalysis. Stimulation with cAMP caused a decrease in cellular Cl and K content in cells grown on impermeable as well as permeable supports. In cells grown on plastic coverslips or Transwell inserts stimulation with cAMP caused a significant decrease of the Na concentration, but no significant effect on the intracellular Na was seen on cells grown on grids. cAMP had no significant effect on the intracellular Ca concentration.

Optimization And Application Of Jet-Freezing, T. Müller, S. Moser, M. Vogt, C. Daugherty, M. V. Parthasarathy

Scanning Microscopy

Cryofixation is considered to be the best method for immobilizing biological material in its natural state. In jet-freezing, the specimen typically is sandwiched between two carriers and kept in place while a coolant is moved very rapidly against the opposite surfaces. The JFD 030 jet-freezing device has been used to optimize the operating parameters. The course of the temperature in place of a specimen was measured with thermocouples and recorded by an IBM-compatible personal computer using a specifically developed software program. Mean cooling rates, over the temperature range of 273K to 173K, achievable with different cryogens, including the non-flammable HCFC …

Scanning Electron Microscopy Of High-Pressure-Frozen Sea Urchin Embryos, Paul Walther, Ya Chen, Marek Malecki, Sara L. Steffen Zoran, Gerald P. Schatten, James B. Pawley

Scanning Microscopy

High-pressure-freezing permits direct cryo-fixation of sea urchin embryos having a defined developmental state without the formation of large ice crystals. We have investigated preparation protocols for observing high-pressure-frozen and freeze-fractured samples in the scanning electron microscope. High-pressure-freezing was superior to other freezing protocols, because the whole bulk sample was reasonably well frozen and the overall three-dimensional shape of the embryos was well preserved. The samples were either dehydrated by freeze-substitution and critical-point-drying, or imaged in the partially hydrated state, using a cold stage in the SEM. During freeze-substitution the samples were stabilized by fixatives. The disadvantage of this method was …

Micro-Pixe (Particle Induced X-Ray Emission) Analysis Of Aluminum In Rat-Liver Using Mev Heavy Ion Microprobes, Yuji Horino, Yoshiaki Mokuno, Atsushi Kinomura, Kanenaga Fujii, Sakae Yumoto

Scanning Microscopy

Heavy ion microprobes (HIM) such as 3 MeV Si²⁺ and 3 MeV p²⁺ have been applied to the elemental analysis by PIXE (proton-induced X-ray emission). It was found that silicon and phosphorus microprobes have several times higher sensitivity for aluminum Kα X-rays than 2 MeV proton microprobes, and detection limits were more favorable in a phosphorus microprobe. Using a 3 MeV p²⁺ microprobe, the liver of a rat, which had been injected with aluminum-lactate, was investigated and it was found that aluminum segregates in areas with a dimension of about 10 μm. These areas could hardly be …

Intracellular And Extracellular Elemental Composition Of The Endolymphatic Sac Studied By X-Ray Microanalysis, Romuald Wroblewski

Scanning Microscopy

X-ray microanalysis was performed along with light microscopy (LM) on rapidly frozen and cryo-sectioned endolymphatic sac tissues of adult guinea pigs, to determine the elemental composition of the different cell types in this tissue as well as the content of the sac lumen. The morphological preservation and spatial resolution of cryo-sectioned endolymphatic sac was found adequate for the identification of the different cell types of the sac in the transmission electron microscope. Further cell type identification was performed by comparing scanning transmission electron microscopy images with LM images on adjacent serial sections. X-ray microanalysis demonstrated differences between epithelial and sub-epithelial …