Biology Commons^™

Three-Dimensional Mapping Of Mineral Densities In Carious Dentin: Theory And Method, J. H. Kinney, G. W. Marshall Jr., S. J. Marshall

Scanning Microscopy

X-ray tomographic microscopy (XTM), a three-dimensional X-ray imaging technique, has been used to quantitatively map mineral concentrations in carious dentin. Data analysis from the XTM study indicates that variations in the mineral concentration surrounding the caries can be imaged in three dimensions with a spatial resolution that is sufficient to detect calcified and enlarged tubule spaces in the lesion. A three-dimensional image of the subsurface lesion indicates that lesion penetration is along the direction of the tubules. The mineral concentration in the uninfected dentin was measured by the XTM to be 1.29 ± 0.14 g/cm³ based upon the tabulated …

A New Versatile System For Freeze-Substitution, Freeze-Drying And Low Temperature Embedding Of Biological Specimens, H. Sitte, L. Edelmann, H. Hässig, H. Kleber, A. Lang

Scanning Microscopy

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 I Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN_2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. FS …

Quantitative Biological Electron Probe Microanalysis With A Wavelength Dispersive Spectrometer, A. Pogorelov, V. Pogorelova, N. V. Repin, I. Mizin

Scanning Microscopy

This paper describes the details of quantitative electron probe microanalysis (EPMA) performed with a wavelength dispersive spectrometer (WDS). EPMA was carried out on the giant neuron of a fresh frozen ganglion from the snail Lymnaea stagnalis. The freeze-dried cryosections were compared with sections of freeze-dried, embedded tissue. It was found, that in the ganglion there are two kinds of neurons with a different chlorine concentration of 11 mmole/liter and 32 mmole/liter. Isolated neurons in culture were shown to differ in elemental composition from those in the ganglion tissue.

Is There A Cell-To-Cell Contact Effect On The X-Ray Dose-Survival Response Of Mammalian Cells?, Nandanuri M. S. Reddy, Krystyna A. Cieszka, Samuil Rozenblyum, Christopher S. Lange

Scanning Microscopy

While a cell-to-cell contact effect has been reported for a Chinese hamster subline V79-171B, this was not observed for another subline V79 171-S. Therefore, we tested whether the cell-to-cell contact effect on cell survival depended on the cell line or the experimental conditions used. We have cultured and compared both sublines under identical conditions. Both sublines, cultured in Eagle's minimal essential medium (MEM) with 15% serum, bad nearly identical cell doubling times and radiosensitivities. For both sublines, the survival of spheroid and monolayer cells subcultured immediately after irradiation were nearly the same, i.e., a radio-protective contact effect for spheroid cells …

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. …

An Experimental Scanning Electron Microscopic Study Of Human Cerebellar Cortex Using The T-Butyl Alcohol Freeze-Drying Device, Teruyuki Hojo

Scanning Microscopy

Specimen preparation methods are very important in scanning electron microscopy (SEM) of nerve tissues. In the present study, a t-butyl alcohol freeze-drying device was used to prepare cerebellar cortex of the human and that of the rat at 15°C and 160 mm Hg. This method has been previously used with success in the preparation of other tissues, such as pancreas and trachea. Relatively large specimens (about 10 mm x 15 mm x 1 mm) of formalin-fixed human and glutaraldehyde-Millonig buffer perfused (1 hour) Wistar rat were rinsed in water, dehydrated in a series of ethanols, immersed in t-butyl …

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 …

Conventional And High Resolution Scanning Electron Microscopy And Cryofracture Techniques As Tools For Tracing Cerebellar Short Intracortical Circuits, Orlando J. Castejón, Robert P. Apkarian, Consuelo Valero

Scanning Microscopy

The present paper shows the potential contribution of conventional and high resolution scanning electron microscopy (SEM) to trace short intracortical circuits in cryofractured fish, primate and human cerebelli. Conventional SEM slicing technique allowed us to identify afferent mossy and climbing fibers and their synaptic relationship in the granular layer. SEM freeze-fracture method exposed the mossy glomerular synapses and the axo-dendritic connections of climbing fibers. At the Purkinje cell layer, the cryofracture process removed the satellite Bergmann glial cell layer, displaying a partial view of the supra-and infra-ganglionic plexuses of Purkinje cells and the ascending pathways of climbing fibers. High resolution …

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 …

Quantitative Dark-Field Mass Analysis Of Ultrathin Cryosections In The Field-Emission Scanning Transmission Electron Microscope, S. Brian Andrews, Roger A. Buchanan, Richard D. Leapman

Scanning Microscopy

The availability of a cryotransfer stage, highly efficient electron energy loss spectrometers, and ultrathin-window energy-dispersive x-ray spectrometers for the VG Microscopes HB501 field-emission scanning transmission electron microscope (STEM) provides this instrument with the potential for high resolution biological microanalysis. Recent technical advances offer cryosections that are thin enough to take advantage of the analytical capabilities of this microscope. This paper first discusses the quantitative characterization of freeze-dried, ultrathin cryosections of directly frozen liver and brain by low-dose dark-field STEM imaging. Such images reveal high-quality sections with good structural detail, mainly due to reduced preparation artifacts and electron beam damage. These …

Multivariate Statistical Analysis Applied To X-Ray Spectra And X-Ray Mapping Of Liver Cell Nuclei, Carmen Quintana, Noël Bonnet

Scanning Microscopy

Principal Components Analysis (PCA) and Factorial Analysis of Correspondence (FAC), two Multivariate Statistical Analyses (MSA), were applied to the analysis of X-ray data. MSA are descriptive methods which graphically display the correlations and anticorrelations between a large number of elements. Series of X-ray spectral data and X-ray maps obtained from rat liver were analyzed with reference to the diffusible elements Na, Mg, Cl, K and Ca and also P and S.

By using an in situ precipitation method, the pyroantimonate method, it was found that the free, precipitable cations Na⁺, Mg²⁺, and Ca²⁺ are, in …

Analysis Of High Quality Monatomic Chromium Films Used In Biological High Resolution Scanning Electron Microscopy, Robert P. Apkarian

Scanning Microscopy

During the recent employment of field emission (FE) in-lens scanning electron microscopes (SEMs), refractory metal deposition technology has co-evolved to provide enhanced contrast of 1-10 nm hydrocarbon based biological structures imaged at high magnifications (> 200,000 times). Pioneer development employing the Penning sputter system in a high vacuum chamber proved that imaging of chromium (Cr) coated biological specimens contained enriched secondary electron (SE)-(I) contrasts. Single nanometer size fibrillar and particulate ectodomains within the context of complex biological membranes were accurately imaged without significant enlargement using the high resolution SE-I mode (HRSEM). This paper reports the transmission electron microscopy (TEM) testing …

The Preparation Of Cultured Cells For X-Ray Microanalysis, Alice Warley

Scanning Microscopy

X-ray microanalysis of cells in culture is being used increasingly for the study of relationships between element (ion) content and cell function. There is, however, no one single method which can be used for the preparation of all different cell types for study by microanalysis. Cells in suspension are usually concentrated by centrifugation, before cryofixation, cryosectioning, and freeze drying. On the other hand cells grown as monolayers are more often studied as whole cell mounts, which are washed to remove the external medium before cryofixation and freeze drying. The alternative approach, sectioning of cell monolayers is rarely used. Some of …

Scanning Electron Microscopic Examination Of The Putative Olfactory Structures Possessed By The Phorid Fly, Megaselia Halterata (Diptera, Phoridae), R. M. Pfeil, R. A. Walsh, R. O. Mumma

Scanning Microscopy

The antennae, palps, and mouth parts sheath of Megaselia halterata (Wood) (Diptera, Phoridae) were examined by scanning electron microscopy to locate putative olfactory sensilla (POS). Most POS were found on the third antenna! segment (Johnston's organ). These POS included a lanceolate peg, a bulbous peg, and two types of pit sensilla. Female flies could be distinguished from males by the predominance of the lanceolate peg on the female Johnston's organ. One type of pit sensillum with a single exterior opening was located below the cuticular surface and housed several pegs. The other type of pit sensillum was domed with a …

The Microvasculature Of Human Infant Oral Mucosa Using Vascular Corrosion Casts And India Ink Injection Ii. Palate And Lip, Q. X. Yu, K. M. Pang, W. Ran, H. P. Philipsen, X. H. Chen

Scanning Microscopy

The microvasculature of human hard and soft palate and lip originating from four infant males and six females, aged 6 months to 2 years was studied by scanning electron microscopy of vascular corrosion casts and light microscopy of India ink injected specimens. The capillary loops of the hard palate mucosa and vermilion border of the lips were found to be tall, numerous and consisted of primary, secondary and tertiary loops. Those of the soft palatal and labial mucosa were short, few in number and demonstrated a simple hair-pin shape originating directly from the subpapillary vascular network. It was concluded that …

The Microvasculature Of The Larynx: A Scanning Electron Microscopic Study, Peter Franz, Seyedhossein Aharinejad

Scanning Microscopy

In ten adult guinea pigs, the microvascular architecture of the larynx was evaluated using microvascular corrosion casts and scanning electron microscopy. The vocal cords were provided with a subepithelial capillary network. The capillaries, freely anastomosing with each other, were supplied and drained via strongly undulating arteries and veins. The undulation of the latter vessels may explain their adaptability to volume changes of the larynx during phonation. The vasculature of the internal perichondrium of the thyroid cartilage was interrupted at the anterior commissure where an avascular zone was present at the origin of the vocal cords. This avascular area is common …

Ultrastructural Changes Of The Vestibular Sensory Organs After Streptomycin Application On The Lateral Canal, Kwang-Sun Lee, Robert S. Kimura

Scanning Microscopy

Early changes in the vestibular sense organs resulting from the application of a streptomycin sulfate soaked Gelfoam® pledget on the fenestra of the lateral semicircular canal were studied by transmission and scanning electron microscopy. Three days after the application, lesions were present in the central part of the lateral crista. The type I sensory cells were more affected than the type JI cells. These sensory cells showed mitochondrial swelling, cytoplasm protrusion at the cell apex, inclusion of multiple vacuoles, fusion or loss of stereocilia, and pyknotic nuclei. Seven days after the drug application, the sensory cell damage extended to all …

Light Element X-Ray Microanalysis In Biology, A. T. Marshall

Scanning Microscopy

It is shown that both qualitative and quantitative light element X-ray microanalysis of biological samples is feasible. These analyses were carried out using ultrathin window (UTW) detectors. Quantitative analysis yields a total element analysis with H estimated by difference or "guesstimated". Comparison with calculated concentrations, or concentrations obtained by chemical analysis, shows that X-ray microanalysis of sections, by the peak to continuum ratio model, give sufficiently accurate results for biological purposes.

The measurement of O concentrations to yield water content is carried out using x-ray imaging techniques, so that the distribution of heavier elements can be spatially related to water …

Improvements In Biological X-Ray Microanalysis: Cryoembedding For Specimen Preparation And Multivariate Statistical Analysis For Data Interpretation, C. Quintana, N. Bonnet

Scanning Microscopy

For biological X-ray microanalysis, cryoembedding (CE) combined with cryofixation (CF) and cryodehydration (CD) was already proposed as an alternative method to freeze-dried cryosections in 1984 by Wroblewski and Wroblewski. CD by freeze-drying (FD) is usually recommended because it provides better retention of diffusible elements. CD by freeze-substitution (FS) has the advantage of being simpler, giving more reproducible preservation of ultrastructure and causing fewer problems for resin infiltration. We have increased the retention of diffusible elements by using home-made devices for CS and CE in the new Lowicryl KllM and HM23 resins. These resins allow samples to be kept at a …

Effect Of Urinary Macromolecules And Chondroitin Sulphate On Calcium Oxalate Crystallization In Urine, A. L. Rodgers, D. Ball, W. Harper

Scanning Microscopy

After filtration and ultrafiltration (10 kD) of 24 hour urine specimens from 12 healthy male subjects, calcium oxalate crystallization was induced in the filtered (FILTD) and ultrafiltered (UF) fractions by administration of a sodium oxalate load. In addition crystallization was also induced in UF fractions to which physiological quantities of chondroitin sulphate (CHON) had been added (UF + CHON). The rate of calcium oxalate crystallization was determined by measuring the rate at which turbidity increased. Crystal numbers and sizes were measured with a Malvern particle size analyzer and by scanning electron microscopy.

Crystallization rates, crystal numbers and crystal sizes were …

Critical Point Mounting Of Kinetoplast Dna For Atomic Force Microscopy, T. Thundat, R. J. Warmack, D. P. Allison, K. B. Jacobson

Scanning Microscopy

Atomic force microscope (AFM) images of intact kinetoplast DNA were obtained from samples prepared utilizing critical point drying. These images are compared with AFM images obtained using conventional methods for DNA deposition. Although the images obtained on chemically pretreated mica show more details than on unmodified mica, images obtained with critical point drying were superior. Kinetoplast networks with expected sizes and structures were routinely observed with critical point drying. The resolution of individual strands of DNA was greatly improved, and image artifacts associated with air dried samples were eliminated. Samples prepared using mildly sonicated kinetoplast DNA show isolated minicircles.

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 …

Signal And Image Manipulation In Microanalysis, P. W. Hawkes

Scanning Microscopy

Scanning electron microscopes, and transmission instruments equipped for EELS, generate a host of signals and hence of images from each pixel of the specimen. Numerous ingenious ways of coping with this multiplicity of information, which may be very different in character, have been devised, but no detailed study has yet been made of the appropriate mathematical structure, with the aid of which all this information could be manipulated reasonably easily.

One such structure falls within the subject that has come to be known as Image Algebra, the principal attraction of which is that we deal directly with entire images …

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 …

X-Ray Microanalysis Of Endocrine, Exocrine And Intestinal Cells And Organs In Culture: Technical And Physiological Aspects, Romuald Wróblewski, Joanna Wroblewski

Scanning Microscopy

In the present study methods for preparation of cultured cells and organ cultures for analytical electron microscopy are investigated. These methods allow qualitative and quantitative analysis of mobile ions in combination with biochemical or morphological studies. Cultured cells can be easily prepared for analytical microscopy and therefore use of in vitro systems for X-ray microanalysis has increased over the last few years. Two major, anhydrous preparation techniques, by which loss or redistribution of ions is minimized, were used: (1) Cells were cryosectioned and analysis carried out on freeze-dried sections obtained from frozen cell monolayers, pelleted cells or organ cultures. (2) …

Microvascular Pattern Of The Retina In The Japanese Monkey (Macaca Fuscata Fuscata), Shigenori Okada, Yoshikuni Ohta

Scanning Microscopy

The microvascular architecture of the retina in the Japanese monkey was revealed using scanning electron microscopy of microvascular corrosion casts.

At the optic nerve disc, the central retinal artery radiated to four primary retinal arterioles: the superior and inferior temporal arterioles and superior and inferior nasal arterioles. A dense retinal capillary network showed regional differences in density. The capillary networks observed were divided in four parts: the optic nerve disc, macula, equator, and ora serrata. At the optic nerve disc, the network was a multi-laminar structure containing the radial peripapillary capillaries in parallel with the nerve fibers at the innermost …

Plasmon Effects In The Particle-Induced Kinetic Electron Emission From Solids, Max Rösler

Scanning Microscopy

Starting from a microscopic description of the particle-induced kinetic electron emission based on the transport equation formalism, the role of different excitation and scattering mechanisms is investigated. Especially, the excitation of electrons by decay of plasmons generated by the impinging particle results in an important contribution to the electron yield. In the case of ion-induced kinetic electron emission, the special features of the energy distribution of emitted electrons are related to the plasmon damping and the plasmon dispersion. In order to describe the available experimental results on nearly-free-electron metals (Al,Mg) in a consistent way, plasmon effects must be taken into …

Induction Of Selective Inner Hair Cell Damage By Carboplatin, S. Takeno, R. V. Harrison, R. J. Mount, M. Wake, Y. Harada

Scanning Microscopy

Carboplatin (diammine [1,1 cyclobutane dicarboxylato (2)-0,0'] platinum) is an anti-cancer agent which can be toxic to the inner ear. We have explored the nature of this ototoxicity in the chinchilla. In this species, initial degenerative changes appear to be restricted to the inner hair cell (IHC) regions of the organ of Corti. This finding is intriguing and unusual since all other known ototoxic drugs, such as aminoglycosides, are predominantly associated with outer hair cell damage.

In the present study, the mechanism of ototoxicity was investigated by comparing two different routes of carboplatin administration. Carboplatin was administered either intravenously (i.v.) or …

Optimal Freeze-Drying Of Cryosections And Bulk Specimens For X-Ray Microanalysis, L. Edelmann

Scanning Microscopy

Electron microscopic investigations of rapidly frozen specimens of striated muscle, either frozen-hydrated or obtained after different dehydration procedures, have shown that the subcellular distribution of the main cellular cation K⁺ or its surrogates Rb⁺, Cs⁺, or Tl⁺ does not follow the water distribution but follows certain proteins. Conflicting results obtained by X-ray microanalysis of freeze-dried cryosections are explained by showing that freeze-drying of bulk specimens and cryosections must be carried out for rather long periods at low temperature in order to avoid severe shrinkage and ion redistribution artefacts. Proposals for future freeze-drying studies are …

Ultrastructural Alterations Of Golgi Apparatus In The Nerve Cells Of Cerebral Cortex In Human Hydrocephalus. A Qualitative Study Using Cortical Biopsies, Orlando J. Castejon, Melecio Diaz, Consuelo Valero

Scanning Microscopy

Cortical biopsies of 17 patients with clinical diagnosis of hydrocephalus and associated pathology were examined in the transmission electron microscope. Moderately and severely edematous neurons showed relevant structural changes of the Golgi apparatus consisting of either discrete or marked dilation, fragmentation, and partial disappearance of Golgi stacked cisternae. In Arnold-Chiari malformation small Golgi complexes of vesicular type and atrophic changes were observed in severely edematous neurons. The microtubules appeared intact, suggesting differential response between Golgi complex and microtubules. Atrophic changes of the Golgi complex coexisted with degenerated presynaptic endings.