Life Sciences Commons^™

Simultaneous Identification Of A Specific Gene Protein Product And Transcript Using Combined Immunocytochemistry And In Situ Hybridization With Non-Radioactive Probes, Gwen V. Childs

Scanning Microscopy

Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory …

Nucleic Acid Detection By In Situ Molecular Immunogold Labeling Procedures, Marc Thiry

Scanning Microscopy

We have recently combined immunogold labeling procedures with molecular biology methods to pinpoint the precise locations of nucleic acids in biological material at the ultrastructural level. These new immunocytological approaches involve the incorporation of labeled nucleotides in the nucleic acids present at the surface of ultrathin sections prior to immunogold labeling. The antibodies used recognize a nucleoside analogue (bromodeoxyuridine) or a hapten (biotin) employed to label nucleotides. Examples of high-resolution detection include DNA or RNA present in different substructures of cell nuclei, and in particular, in adenovirus-induced intranuclear regions of HeLa cells. In addition to being highly sensitive and specific, …

A Comparison Of The Renal Structures Of The Anaconda And The Ball Python, H. Ditrich

Scanning Microscopy

The renal vascular system of the Ball python (Python regius) and the anaconda (Eunectes noteus; Serpentes - Squamata) has been investigated using light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) of vascular corrosion casts and critical-point dried non-corroded specimens. The average glomerular diameters of these two species differ significantly (anaconda: 59.1 μm, python: 124.3 μm). Also, the relative proportions of the renal tubules are different. These findings can be related to the different habitats of the two species (aquatic versus terrestrial environment).

Labeling With Nanogold And Undecagold: Techniques And Results, James F. Hainfeld

Scanning Microscopy

A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and allow many new probes to be made with peptides, nucleic acids, lipids, drugs, and other molecules. A new fluorescent-gold conjugate is useful for examining localization by fluorescence microscopy, then visualizing the same label at the ultrastructural level in the electron microscope.

Lectin-Cytochemistry Of Experimental Rat Nephrolithiasis, W. C. De Bruijn, R. De Water, E. R. Boeve, P. R. W. A. Van Run, P. J. Vermaire, P. P. M. C. Van Miert, J. C. Romijn, C. F. Verkoelen, L. C. Cao, F. H. Schroder

Scanning Microscopy

Lectin reactivity in epithelial apical cell coats of normal rat kidneys was compared to that from animals subjected to crystal inducing diets (CID). The aim was to see whether the absence of lectin reactivity in cell coats is related to intratubular calcium oxalate crystal retention. In normal rat kidneys, after a pre-embedding procedure, it was observed that at the ultrastructural level, reactivity was present but that the lectin specificity for the various parts of the nephron might have to be reconsidered. There was heterogeneity between the epithelial cells with respect to the presence of coat material in the tubular cell …

Effects Of Ionizing Radiation On Cartilage: Emphasis On Effects On The Extracellular Matrix, M. Cornelissen, H. Thierens, L. De Ridder

Scanning Microscopy

In this report, we review data dealing with radiation effects on cartilage. More specifically, we emphasize on alterations caused in the extra-cellular cartilage matrix. Although radiation studies predominantly describe the effect on the structure of DNA and on the mitotic activity of cells, alterations caused by the effect on the non-mitotic activity can also be important. Cartilage, having an extracellular matrix composed of 2 major components, aggrecan and collagen, provides a good model to study this kind of radiation effects. The following topics concerning literature data are summarized: effects on the amount of matrix synthesized, effects on the activity of …

Comparative Scanning, Transmission And Atomic Force Microscopy Of The Microtubular Cytoskeleton In Fenestrated Liver Endothelial Cells, Filip Braet, Ronald De Zanger, Wouter Kalle, Anton Raap, Hans Tanke, Eddie Wisse

Scanning Microscopy

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and …

Quantitative Imaging In Electron And Confocal Microscopies For Applications In Biology, N. Bonnet, L. Lucas, D. Ploton

Scanning Microscopy

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be …

Photoemission And Free Electron Laser Spectromicroscopy: Photoemission At High Lateral Resolution, G. Margaritondo

Scanning Microscopy

The move of photoemission analysis from the macroscopic to the microscopic domain has been accelerated by the advent of new ultrabright synchrotron sources of soft-X-rays. This makes an overview of photoemission spectromicroscopy, photoemission at high lateral resolution, quite timely. The overview begins with the basic concepts and problems, both technical and of data-taking strategy. Then, it presents a small number of examples of results in physics and biology, such as local chemical fluctuations in superconductors, semiconductor interfaces and the microchemistry of biological systems. The presentation includes the first experimental results from two new ultrabright synchrotron facilities: ELETTRA (in Italy) and …

Different Approaches To The Study Of Apoptosis, E. Falcieri, L. Stuppia, A. Di Baldassarre, A. R. Mariani, C. Cinti, M. Columbaro, L. Zamai, M. Vitale

Scanning Microscopy

The morphological features of cell undergoing programmed cell death is well known and has been widely described in a number of experimental models with a variety of apoptotic triggering agents. Despite the similar cell behaviour, underlying molecular events seem variable and only partially understood. A multiple approach appears crucial to better clarify the phenomenon. The first technique, DNA gel electrophoresis, allows the identification of fragmented DNA and has been long considered the hallmark of apoptosis. Different patterns of DNA cleavage, which can be identified by conventional or "pulsed-field gel" electrophoresis, are presented and discussed. "In situ" labelling methods are also …

X-Irradiation-Induced Disorganization Of Cytoskeletal Filaments And Cell Contacts In Ht29 Cells, Z. Somosy, M. Sass, G. Bognar, J. Kovacs, G. J. Koteles

Scanning Microscopy

Organization of cytoskeleton and cell contacts were studied by immunochemistry and electron microscopy in confluent HT29 cultured cells following exposure to 0.5 and 1.0 Gy doses of X-ray. Microtubules were resistant to irradiation, whereas, the actin and intermediate filaments disrupted rapidly following the treatment and their components appeared as clumps of actin and cytokeratin aggregates in the cytoplasm as demonstrated by immunochemistry. Loss of cell contacts and decrease in the number of desmosomes was also characteristic of irradiated cells. Electron microscopy revealed intact desmosomes in control cells and abnormal desmosomes in the irradiated samples characterized by the absence of tonofilaments. …

Use Of Colloidal Gold And Neutron Activation In Correlative Microscopic Labeling And Label Quantitation, B. J. Darien, P. A. Sims, K. T. Kruse-Elliott, T. S. Homan, R. J. Cashwell, A. J. Cooley, R. M. Albrecht

Scanning Microscopy

Albumin was conjugated to 16 nm gold particles (Alb-Au16) and infused into anesthetized pigs to determine if plasma, tissue and bronchoalveolar lavage (BAL) fluid concentrations of gold could be quantitated by neutron activation (Au198). Additionally, transmission electron microscopy (TEM) of lung and liver samples was evaluated for sites of gold distribution and morphological changes. The minimum concentration of gold detected by neutron activation ranged between 1.4 and 1.9 ppb (ng/gm of sample). No gold was detected in the plasma of pigs prior to Alb-Au16 infusion, while mean post infusion concentrations were 1.037 ± 0.69 ppm (μg/gm plasma, ±SD). The concentrations …

The Effect Of Arf-Excimer Laser Irradiation Of The Human Enamel Surface On The Bond Strength Of Orthodontic Appliances, U. Stratmann, K. Schaarschmidt, M. Schürenberg, U. Ehmer

Scanning Microscopy

This study investigated enamel laser conditioning as an alternative to acid etching in bracket therapy. In preliminary experiments optimal laser parameters for achieving a bond strength of 6-10 N/mm² were defined. Enamel surface morphology was assessed and the ablation depth was measured on serial enamel sections. Thirty human molars were exposed to 193 nm ArF-excimer laser radiation (energy density: 260 mJ/cm²) by single pulse application of 23 nanoseconds. Thirty molars were etched with phosphoric acid (37%) for 60 seconds. The brackets from the treated molars and 30 untreated molars were debonded vertically for tensile bond strength measurement. …

Ultrastructural Patterns Of Cell Damage And Death Following Gamma Radiation Exposure Of Murine Erythroleukemia Cells, R. Di Pietro, E. Falcieri, L. Centurione, M. A. Centurione, G. Mazzotti, R. Rana

Scanning Microscopy

Radiation causes damage to cell surface membranes, cytoplasmic organelles, and the nuclear process of DNA synthesis and repair, and this eventually results in different modes of cell death. In this study we examined murine erythroleukemia (MEL) cells, exposed to 15 and 60 Gy of 10 MeV photonic energy, and left in culture for up to 96 hours. Electron microscopical analysis was performed on conventionally embedded samples and freeze-fracture replicas, in order to detect ultrastructural patterns of cell damage and death. Of interest was the observation of chromatin condensates, nuclear membrane associations and nuclear pore redistribution during early apoptosis. Pronounced rearrangements …

Time-Resolved Cryofixation Methods For The Study Of Dynamic Cellular Events By Electron Microscopy: A Review, Keith P. Ryan, Gerd Knoll

Scanning Microscopy

The preservation of cells for electron microscopy by chemical fixation is a lengthy process, requiring up to 30 minutes for cytoplasmic stabilisation. This time lag enables many changes to occur in specimens so that they may not reflect their living state when they are observed in electron microscopes. Many artefacts can be avoided by using cryofixation, which freezes specimens over a period that is measured in milliseconds, so that specimens are preserved by cryoimmobilisation. This time resolution can be used to study rapid processes in biology and chemistry because, although electron microscopes cannot observe dynamic cellular events directly, processes can …

Intercellular Interactions In Pc12 Cells Overexpressing Beta/A4 Amyloid, Gladys E. Maestre, Barbara A. Tate, Ronald E. Majocha, John Maguire, Charles A. Marotta

Scanning Microscopy

The amyloid precursor protein (APP) is an integral membrane component of eukaryotic cells. A variety of research approaches have addressed the contribution of the β amyloid peptide region of the APP to neuritic plaque structure and formation in the Alzheimer disease brain as well as the relationship between β amyloid accumulation and the occurrence of dementia. However, there is limited information available concerning the cellular consequences of amyloid deposition. The present studies were undertaken to investigate the relationship between β amyloid and intercellular junctions. Transfected PC12 cell lines, that overexpress the β amyloid peptide, exhibit structural and functional alterations at …

Environmental Scanning Electron Microscope Examination Of Paper In High Moisture Environment: Surface Structural Changes And Electron Beam Damage, Päivi Forsberg, Pierre Lepoutre

Scanning Microscopy

Supercalendered and coated papers (SC and LWC) were examined using an environmental scanning electron microscope (ESEM). Moderate structural surface changes were observed as water condensed on the surface in a high moisture environment. The changes were fully or partially reversible depending on the sample origin. A wide range of contact angles could be observed when condensing water on uncoated wood fibers. While there was no visible indication of irradiation damage on the commercial paper samples examined nor on mechanical pulp fibers, attempts to look at chemical pulp fibers during wetting to examine fiber swelling were unsuccessful because of very rapid …

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 …

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, …

Distribution And Intracellular Localization Of Titanium In Plants After Titanium Treatment, G. Kelemen, A. Keresztes, E. Bacsy, M. Feher, P. Fodor, I. Pais

Food Structure

The physiological role of titanium in plants has not been elucidated yet, but a higher rate of growth , greater chlorophyll content and higher productivity, among others , may be attributed to this element. We have investigated the distribution of titanium in wheat seedlings after Titavit (a Ti ·ascorbate containing plant conditioner) treatment applied either through the leaf or through the root. In field experiments, we also sprayed spinach plants with Titavit. We have found a practically unidirectional tran slocation of Ti from the shoot into the root, and a preferential local ization of Ti in the nuclear cell fraction …

Practical Aspects Of Electron Microscopy In Dairy Research, Miloslav Kalab

Food Structure

Milk products are based mostly on casein micelles, fat globules, and whey proteins. The former two constituents are corpuscular and whey proteins become corpuscular when coagulated. Structural changes in these basic constituents during processing have been studied by electron microscopy. This review discusses the structures of yoghurt, curd, cheeses (hard cheeses, mould-ripened cheeses, cream cheeses, and process cheese), cream, milk powders, and nontraditional dairy products. Defects and deviations from traditional structures of these products are explained where the causes are known. Examples of such causes are foaming of milk, presence of unusual ingredients (bacterial polysaccharides, whey protein concentrates), and alterations …

Quantitative And Computer Assisted Electron Microscopic And Microprobe Studies In Dermatology, B. Forslind, A. Emilson

Scanning Microscopy

Electron microscopes are not yet routine instruments in modern dermatological pathology even though they have provided detailed data about pathological changes in the skin for more than three decades. At present, dermatopathology is still dominated by light microscopy and especially so since the introduction of immunological techniques such as the use of monoclonal antibodies. These tools applied at electron microscopic resolution, however, may provide the ultimate identification of cells and subcellular components. In addition, electron microscopes have no peers in areas of quantitative investigation at subcellular levels, e.g. morphometry.

The electron microprobe provides a unique tool in elemental analysis and …

Functional And Morphological Studies Of Mitochondria Exposed To Undecagold Clusters: Biologic Surfaces Labeling With Gold Clusters, E. Valdivia, C. Gabel, J. E. Reardon, C. A. Cajacob, H. Yang, R. S. Wehbi, G. L. Scott, P. A. Frey, L. A. Fahien

Scanning Microscopy

This study reports morphological and functional alterations observed in respiring isolated mitochondria when they are exposed to nonpenetrating, positive electrostatically charged synthetic undecagold clusters. Modification of the undecagold clusters positive charges change or prevent the functional effects and the binding to the outside surface of the mitochondria. The mitochondrial functional alterations are dependent on the oxidative phosphorylation capacity of the isolated organelles. The results of these experiments indicate that artificial undecagold may be useful to explore the molecular mechanisms of biological energy transducers which require electric charges separation, ionic fluxes, and electric surface properties.

Structure Of Rat Kidneys Following Microwave Accelerated Fixation, Jayashree A. Gokhale, Saeed R. Khan

Scanning Microscopy

In contrast to fixation of tissue in externally heated fixative, microwave-irradiation can generate uniform internal heat, which is of utmost importance for successful fixation of biological tissue. To evaluate the effectiveness of microwave-accelerated chemical fixation, we compared the structure of rat kidney fixed by a conventional method and a microwave-accelerated method, by scanning and transmission electron microscopy. Following perfusion, rat kidney pieces 1-2 mm in size were irradiated in Karnovsky's fixative in a domestic Amana microwave oven, till the temperature of the fixative reached 45-50°C. For conventional fixation, tissue pieces were fixed overnight at room temperature in the same fixative. …

Acquisition Hardware For Imaging, Peter Rez, J. K. Weiss, W. J. De Ruijter

Scanning Microscopy

In electron microscopy images can either be recorded in parallel (Transmission Electron Microscopy) or acquired as the variation in a signal as a probe is scanned over the specimen (Scanning Electron Microscopy). To extract the most information from an image requires that the best possible systems are used for acquiring image data. Ultimately, the limit to information capture is achieved when every electron from the scattering event of interest is recorded. The ideal system can be realised both for parallel recording with scientific grade CCD cameras, and for scanning microscopy with single electron counting electronics. The data rates from these …

Application Of The Wavelet Transform To The Digital Image Processing Of Electron Micrographs And Of Backreflection Electron Diffraction Patterns, A. Gómez, L. Beltrán Del Río, D. Romeu, M. Jose Yacamán

Scanning Microscopy

In this work we explore the use of the so-called wavelet transform in the digital image processing of micrographs. The wavelet transform of an image f(x,y) is defined as:

Wf(s,u,v)

= f(x,y) s Ψ(s(x-u),s(y-v)) dxdy

where Ψ is an analyzing function called "wavelet" and which is in our examples always taken to be the "Mexican hat" given by

Ψ(x)=(2-(x²+y²))exp(-(x²+y²)/2)

Some synthetic images are shown in which it can be clearly seen how the wavelet transform can be useful to reveal edges and to emphasize the boundaries of the clusters.

The technique is …

The Application Of Multispectral Techniques To Analytical Electron Microscopy, P. G. Kenny, M. Prutton, R. H. Roberts, I. R. Barkshire, J. C. Greenwood, M. J. Hadley, S. P. Tear

Scanning Microscopy

The York multispectral analytical electron microscope (MULSAM) was the first instrument specifically designed to acquire and process multiple Auger, X-ray, backscattered electron, elastically scattered electron, scanning electron microscopy (SEM) and specimen absorption current images simultaneously. Analyzing combinations of these signals with multispectral correlation techniques yields more information than would normally be obtained by treating each image separately. This paper reports some of the multispectral methods we have investigated at York which may be of use to other workers. Included are (1) a method that corrects for beam current fluctuations during long acquisition runs which is based on the anti-correlation between …

Atomic Resolution Electron Holography, K. Ishizuka, T. Tanji, A. Tonomura

Scanning Microscopy

It has been demonstrated that electron holography is a very powerful tool to investigate an electromagnetic potential in medium resolution, since the phase of an electron wave is approximately proportional to the potential. Now, electron holography is at the second stage of development: to establish holography at atomic resolution and further to realize Gabor's idea to improve the resolution restricted by the spherical aberration of the objective lens. We investigate the possibility of electron holography to get information at atomic resolution by computer simulations as well as by digital processing of electron holograms. We show that the phase distribution has …

Phase Space Methods In Image Formation Theory, Victor Castaño, Gustavo Vásquez-Polo, Ramón Gutiérrez-Castrejón

Scanning Microscopy

A review of the basic mathematical principles and properties of some phase space functions (Wigner and Ambiguity functions) is first presented in a more general mathematical context. Then, we deal in detail with three applications : image formation in optical systems (specifically cascading systems of lenses), image formation in Transmission Electron Microscopy, by taking advantage of some formal similarities with the previous examples and, finally, contrast transfer functions in Electron Microscopy in order to obtain 2-dimensional plots which provide information on the behavior of a transmission electron microscope. Also, the relations of these functions with other mathematical tools utilized in …

Segmentation Of Pores In Backscattered Images Of Sediments And Soils And Their Relationship To Domain Structure, M. W. Hounslow, N. K. Tovey

Scanning Microscopy

A new technique to quantify variations in pore structure across an image is described in this paper. The method involves several stages as the original images are rarely suitable for simple segmentation by the selection of a single threshold. The processing first involves the application of a Wiener filter to remove imaging artefacts and to sharpen the edge of void/particle contacts. This image reconstruction is followed by the selection of a global threshold based on the relative contrast method, to create a binary image separating the voids from the particles. Such an image can be used to evaluate pore structure …