Biology Commons^™

Restricted Energy Transfer In Laser Desorption Of High Molecular Weight Biomolecules, Akos Vertes, Renaat Gijbels

Scanning Microscopy

Producing ions from large molecules is of distinguished importance in mass spectrometry. In our present study we survey different laser desorption methods in view of their virtues and drawbacks in volatilization and ion generation. Laser induced thermal desorption and matrix assisted laser desorption are assessed with special emphasis to the recent breakthrough in the field (m/z > 100,000 ions produced by matrix assisted laser desorption). Efforts to understand and describe laser desorption and ionization are also reported. We emphasize the role of restricted energy transfer pathways as a possible explanation to the volatilization of non-degraded large molecules.

Retinal Changes In A Mutant Form Of Goldfish With Megalophthalmia, David T. Yew, Winnie W. Y. Li, C. Au, H. L. Choi, Q. D. Yang, Patrick K. H. Chan

Scanning Microscopy

The retinal changes of a mutant strain of goldfish with megalophthalmia were studied by histology, electron microscopy and biochemistry. Changes in the morphology of the pigment epithelium, decrease in number of photoreceptors, thinning out of retinal layers and the existence of spaces in the retina were all features as the eyes grew in size. Invasion of macrophages was also evident in the retina. A decrease in leucine uptake per milligram of retina was also detected as the eye grew beyond 0.8 millilitre in volume. These changes, although related to volume changes (i.e., growth) of the eyes, were found to have …

The Primary Energy Dependence Of Backscattered Electron Images Up To 100 Kev, I. Geuens, B. Nys, J. Naudts, R. Gijbels, W. Jacob, P. Van Espen

Scanning Microscopy

The backscattered electron coefficient is known to be primarily dependent on the atomic number of the sample. If the atomic number increases, the backscattered electron coefficient increases, which results in a higher intensity in the backscattered electron image. The dependence of the primary electron energy is somewhat more complicated. Using photographic material (with composition AgBr-AgI), it is seen that the contrast in the backscattered electron image increases with the primary electron energy. Using three independent methods, based on image analysis techniques, it is shown that the difference between the backscattered electron coefficient of AgBr and AgI increases with the primary …

Theodore Alvin Hall: A Biographical Sketch And Personal Appreciation, Brij L. Gupta

Scanning Microscopy

As the first in a series of two papers contributed on the occasion of a special program held at the Scanning Microscopy/1989 meeting in Salt Lake City in honour of Dr. T.A. Hall, and in recognition of his achievements for biological electron probe X-ray microanalysis, this paper provides a biographical sketch of Dr. Hall, as well as a bibliography.

Growing And In-Situ Processing Of Cells On Beem Capsule Caps For Scanning Electron Microscopy, Eugene F. Bernard, Tatsuo Hase

Scanning Microscopy

A simple technique for processing cells grown on BEEM capsule caps for scanning electron microscopic (SEM) observation is described. The cells adhere to a substratum on the inner surface of caps coated with either an egg white-polyvinylpyrrolidone solution or a polylysine solution in the case of eukaryotic cells and an agar solution in the case of bacteria. The adhering cells are cultured and then processed in situ for SEM observation. Electron micrographs revealed details of the shape of the cells growing on the surface of the substrate. The technique seems to be particularly well suited for observing sequential morphological changes …

Ultrastructure Of Coronary Arterial Endothelium In Atherosclerotic Swine Suggests Lipid Retro-Endocytosis, Keith A. Robinson, Robert P. Apkarian

Scanning Microscopy

During experimental atherosclerosis, arterial endothelial cells show characteristic ultrastructural changes including the appearance of increased numbers of plasmalemmal and cytoplasmic vesicles. These structures have been shown by tracer studies to participate in the transcellular transport of low-density lipoprotein cholesterol and β-very-low-density lipoprotein cholesterol from the arterial lumen into the abluminal extracellular matrix. Although this probably represents the major lipid transport pathway, other forms of transport may exist. We document the presence of apparent lipid structures averaging approximately 300 nm in diameter at or near the luminal surface of coronary arterial endothelium of atherosclerotic miniature swine. The structures exhibited a particulate …

Scanning Electron Microscopy Of The Forming Enamel Of Rat Incisor: Influence Of Fixative And Treatments Interacting With The Organic Matrix, J. P. Salomon, J. M. Legrand, M. Goldberg

Scanning Microscopy

The forming enamel of rat incisor was either treated before fixation with one of these five treatments: ultrasonication, NaOCl, chloroform/methanol, NaOH or tween 20; or the incisors were first fixed and then submitted to one of these treatments. The structure of the forming enamel, compared with sections fixed with aldehyde and submitted to osmic maceration, depends on the way the samples were treated and on the fixative used. An amorphous material, removed by osmium tetroxide but not by aldehyde solution was seen to be located between fiber-like structures. In the region of inner enamel secretion the thickness of interrod septa …

Ted Hall And The Science Of Biological Microprobe X-Ray Analysis: A Historical Perspective Of Methodology And Biological Dividends, Brij L. Gupta

Scanning Microscopy

This review surveys the emergence of electron probe X-ray microanalysis as a quantitative method for measuring the chemical elements in situ. The extension of the method to the biological sciences under the influence of Ted Hall is reviewed. Some classical experiments by Hall and his colleagues in Cambridge, UK, previously unpublished, are described; as are some of the earliest quantitative results from the cryo-sections obtained in Cambridge and elsewhere. The progress of the methodology is critically evaluated from the earliest starts to the present state of the art. Particular attention has been focused on the application of the method in …

Three-Dimensional Morphological Analysis Of Nerve Cells By Scanning Electron Microscopy. A Review, O. J. Castejón

Scanning Microscopy

The present review provides a rational and new approach to study the three-dimensional morphology of nerve cells in situ at both cellular and macromolecular levels, by means of conventional (SEM) and high resolution scanning electron microscopy (HRSEM). The slicing and ethanol-cryofracturing methods, the freeze-fracture SEM method and tissue preparation for HRSEM have been described. Nerve cell outer surface, axon hillock and initial axon segment, axonal collateral ramifications and dendritic processes were visualized either by the slicing technique or the cryofracture method displaying neuronal geometry in situ. The cleavage plane occurred at the satellite neuroglial sheath exposing somatic hidden surfaces of …

Microvillus Form Of Focal Anchorage In Human Chang Liver Cells Rounded By Antiporter Activation: Scanning Electron Microscopy Profiles And Evidence Of Traction Origin, K. H. Sit, B. H. Bay, K. P. Wong

Scanning Microscopy

Na⁺/H⁺ anti porter activation in human Chang liver cells produces a flat-to-round (FTR) change in cell shape with gross reduction in cell profile area. Scanning electron microscopy (SEM) vividly displays a third phenomenon, viz., the development of focal microvillus anchors. Reduction in cell profile area concomitant with the development of this microvillus form of focal anchorage is quantitated by on-line image analysis during SEM examination. The reduction in profile area is corroborated by spectrophotometric digitization in light microscopy. Transmission electron microscopy (TEM) of rounded cells shows large endocytic channels and endosomes consistent with the observation of internalization …

Fluorescence Recovery After Photobleaching Techniques To Measure Translational Mobility In Microscopic Samples, Ken Jacobson, Fen Zhang, Tsong-Tseh Tsay

Scanning Microscopy

The scope of photobleaching applications and the method itself are briefly reviewed. Two current applications in this laboratory are then outlined. First, the use of spatial Fourier transforms to analyze video photobleaching measurements is presented. This method extracts diffusion coefficients using all the image data and it does not require that the initial condition created by photobleaching be known. Second, the use of genetic engineering methods coupled with photobleaching analysis is discussed as means to uncover the structural determinants of membrane protein lateral mobility.

Three-Dimensional Morphology Of Cerebellar Protoplasmic Islands And Proteoglycan Content Of Mossy Fiber Glomerulus: A Scanning And Transmission Electron Microscope Study, O. J. Castejón, H. V. Castejón

Scanning Microscopy

The present review summarizes the outer and inner surface features of mossy fiber glomeruli in vertebrate cerebellar granular layer as seen by conventional scanning electron microscopy (SEM) and SEM freeze-fracture method. The intracortical trajectory of mossy fibers and their synaptic contacts with granule cell dendrites were traced by the slicing and freeze-fracture techniques revealing the radial distribution of granule cell dendrites around the central mossy rosette. The "en passant" nature of mossy fiber synaptic contacts and the participation of Golgi cell axonal ramifications were demonstrated. The results obtained were compared with available light and transmission electron microscopy data. The freeze-etching …

Alpha-Particle Irradiation-Induced Change In Bronchopulmonary Macrophage Morphology, In Vitro, T. M. Seed, G. K. Niiro

Scanning Microscopy

Bronchopulmonary macrophages, isolated from canine lungs by saline lavage and grown in tissue culture for short periods, were acutely irradiated with a range of doses of either Americium-241 alpha particles (0.03-48 Gy) or 250 keV x-rays (0.5-24 Gy). Following a 24-hour reincubation and "expression" period, cells were examined for radiation-induced changes in overall viability, as well as in cell morphology and ultrastructure. Results indicated that neither quality of radiation had much effect on cell viability over dose ranges examined, but substantial changes in cell volume, surface topography, and cytoplasmic features were noted, especially in the alpha-particle-irradiated specimens. Results support the …

A Scanning Electron Microscopic Morphological And Semi-Quantitative Evaluation Of Rat Stomach Treated With Colloidal Bismuth Subcitrate And Alcohol, C. Winters, S. M. Hinsull, Z. Gregory

Scanning Microscopy

Scanning electron microscopy was utilised to study the effect of absolute alcohol on the normal morphology of the rat stomach, together with the gastroprotective actions of colloidal bismuth subcitrate. Studies on normal gastric morphology revealed that the major portion of the stomach was covered by a protective coating of mucus. However, there was considerable variation in the integrity of the mucosal surface of the control animals, with the loss of surface epithelial cells in some regions which may account for the variation in response to necrotising agents. The long-term administration of the gastrocytoprotective agent colloidal bismuth subcitrate resulted in a …

The Response Of The Tracheal Epithelium To Concomitant Cis-Diamminedichloroplatinum (Ii) And Radiation. An Electron Microscopic Study In Rabbits, C. H. Håkansson, M. Albertsson, M. Palmegren, J. Ranstam

Scanning Microscopy

The ciliated epithelium of the rabbit trachea was irradiated with daily fractions of 2 Gy up to an accumulated dose of 20 Gy (total dose: 2, 6, 10, 16, or 20 Gy). Fifteen to forty-five minutes before the start of each irradiation 0.3 mg Cis-diamminedichloroplatinum (cis-DDP) was given by intraperitoneal injection to each rabbit. Examinations were carried out 1-10 days after each fractionation schedule, when specimens were taken for morphological investigations.

Scanning electron microscope (SEM) examination showed a gradual development of ciliary damage, from blebs on the cilia to swollen tips, broken and bent cilia and finally an epithelial injury …

Timed Acid Bath Effects On Kidney Podocytes Prepared For Scanning Electron Microscopy, B. J. Moore, K. R. Holmes

Scanning Microscopy

In perfusion fixation of dog kidney glomeruli, observation of tear fractured surfaces by scanning electron microscopy reveals the deposition of "debris" on the surface of glomerular visceral epithelium (podocytes) that is not deposited on other kidney tissue surfaces. It is of concern to remove this debris without damage to tissue surfaces. A 5 minute bath in 8N HCI (hydrochloric acid) following postfixation in 1% OsO₄ (osmium tetroxide) removes only some of the debris. A 10 minute acid bath removes this debris without visible damage to podocyte surface structures such as cilia, plicae or blebs. A 20 minute acid bath …

Transplacental Cardiotoxicity Of Cocaine: Atrial Damage Following Treatment In Early Pregnancy, Jacques Gilloteaux, John P. Dalbec

Scanning Microscopy

Using light, transmission (TEM) and scanning (SEM) electron microscopy, cocaine-induced defects were observed in hamster atria. Compared with controls, the treated atria from neonates show endocardial and myocardial damages as the atrial walls thicken. SEM micrographs show intensive blebbing, damage and incomplete coverage of myocardium by the endocardial endothelium. TEM data demonstrate blebs, thinning, and other endothelial cell injuries and complement the SEM findings. Areas of endothelial sloughing may facilitate the formation of luminal and mural thrombi as noticed in many neonatal atria. Adjacent subendocardial myocardial cells display contraction bands, swellings, and vacuolizations. Local and large areas of damaged myocardial …

Copper Toxicity In Erwinia Amylovora : An X-Ray Microanalytical Study, N. Hodson, D. C. Sigee

Scanning Microscopy

The effect of Cu²⁺ ions on the growth and elemental composition of the phytopathogenic bacterium Erwinia amylovora was investigated by in vitro culture in nutrient broth supplemented with CuSO₄.

No detectable inhibition in bacterial growth occurred with medium supplemented at 10^-4M Cu (compared to control medium with no added Cu), but partial inhibition occurred at 10^-3M Cu (limiting toxicity) and complete inhibition at 10^-2M Cu.

Under conditions of limiting toxicity, incubation in Cu²⁺ ions leads to a reduction in the general synthesis of cell materials and the formation of abnormally …

An Introduction To Monte Carlo Simulations, David C. Joy

Scanning Microscopy

The interaction of an electron beam with a solid can be modeled by the so-called Monte Carlo method. This technique produces a stepwise simulation of the electron trajectory by using random numbers to predict scattering angles on the basis of theoretical probability distributions or empirical models. The physical basis of electron scattering in a solid is described and two generic types of Monte Carlo model are then developed together with suggested examples of their application. An IBM PC compatible disc containing these programs is available from the author.

Imaging Of Magnetic Microstructures At Surfaces: The Scanning Electron Microscope With Spin Polarization Analysis, H. P. Oepen, J. Kirschner

Scanning Microscopy

The scanning electron microscope with spin polarization analysis of secondary electrons has been proven to be a powerful tool for studying magnetic microstructures. Secondary electrons created at the surface of a ferromagnet are spin-polarized and contain information about the sample magnetization and its orientation. The combination of a spin polarization analyzer with a scanning electron microscope yields an unique apparatus for probing magnetic properties on a very small lateral scale. The magnetic resolution of < 40 nm is demonstrated. This type of microscope provides high magnetic contrast, while the surface morphology is strongly or even totally suppressed. The capability of studying magnetic properties of semi- infinite samples as well as in ultrathin films is demonstrated with a Fe(lOO) single crystal, video tape, CoCr perpendicular recording medium and ultrathin cobalt films.

Effects Of Low Energy Beta-Irradiation From Tritiated Water On The Morphology Of 3t3 Fibroblasts, Z. Somosy, Tamara Kubasova, J. Kovács, G. J. Köteles

Scanning Microscopy

Cellular alterations of cultured 3T3 cells irradiated with beta-rays from tritiated water were studied by scanning and transmission electron microscopy. We observed decreased negative surface charges, vacuolization of rough endoplasmic reticulum and Golgi-complex, degeneration of mitochondria, increase of lysosomal activity and changes in distribution and amount of microfilaments in the irradiated cells, that parallelled changes in cell shape.

Preservation And Immunogold Localization Of Lipids By Freeze-Substitution And Low Temperature Embedding, Wim Voorhout, Ida Van Genderen, Gerrit Van Meer, Hans Geuze

Scanning Microscopy

The success of postembedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level.

In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and …

Diffusion Of Water In Biological Tissues, H. E. Rorschach, C. Lin, C. F. Hazlewood

Scanning Microscopy

A method is presented for obtaining simple approximate solutions for the problem of self-diffusion in an ordered array of obstacles. Our results are compared with some previous exact and approximate solutions, and we find that our method agrees well with the exact results over a large range of the volume fraction of the obstructions. It is shown that there is an important distinction between measurements of the diffusion coefficient by the capillary flow method and the spin-echo method. The modifications for the spin-echo case are given and applied to recent measurements on the anisotropy of the self-diffusion of water in …

Reliability Of Intracellular Water And Ion Distributions As Measured By X-Ray Microanalysis - A Review, Thomas Von Zglinicki

Scanning Microscopy

X-ray microanalysis can be an important tool to reveal the spatial relationships between polyelectrolytes, ions, and water as they occur within cells and tissues in vivo. To reach this goal, at least two of these three closely interrelated variables should be measured independently. Moreover, the absence of systematic errors should be proven.

The present review discusses the probability of artificial ion and water shifts between intracellular compartments due to the growth of dendritic ice crystals much larger than the cross-sectioned remnants commonly seen in frozen-dried sections. Considering the possible mechanism of ice crystal growth it is concluded that ions and …

Cryopreparation Of Tissue For Clinical Applications Of X-Ray Microanalysis, Godfried M. Roomans

Scanning Microscopy

A number of diseases is associated with changes in ion and/or water distribution at the tissue or cell level, and X-ray microanalysis can be used to investigate the relationship between the disease process and the changes in elemental distribution. For analysis of diffusible elements by X-ray microanalysis, the tissue has to be prepared by cryotechniques. To carry out studies of this kind in a clinical environment poses a number of problems. Some of these problems occur already before the tissue is frozen, others are caused by the practical and ethical limitations that are imposed on the freezing method itself when …

Freezing: Facts And Hypothesis, J. Dubochet, K. Richter, H.-V. Roy, A. W. Mcdowall

Scanning Microscopy

Hexagonal ice crystals formed in frozen biological specimens are large and branched. They can produce severe structural damage by solute segregation but there are also cases where they seem to cause only minor damage. When cooling is more rapid, cubic ice crystals can be formed. These are small and in general, they cause little damage. These observations can be readily explained with the hypothesis that large hexagonal ice crystals can originate from the rewarming induced transformation of a large number of cubic ice crystals. This transformation would take place without significant solute displacement.

The Ultrastructure Of Cryo-Sections And Intact Vitrified Cells - The Effects Of Cryoprotectants And Acceleration Voltage On Beam Induced Bubbling, P. M. Frederik, P. H. H. Bomans, M. C. A. Stuart

Scanning Microscopy

Chemically fixed pancreas was infiltrated with various cryoprotectants to obtain homogeneously vitrified samples upon cooling. The suitability of these samples for cryo-ultramicrotomy was tested. Contrast was hardly detectable initially in thin cryo-sections but increased upon irradiation, irrespective of the cryoprotectant (glycerol, propylene glycol, methanol) used. Contrast and beam damage were analyzed in vitrified thin films from collagen, phospholipid vesicles and various concentrations of glycerol. Glycerol increased the beam sensitivity of both collagen and phospholipid vesicles, but diminished the contrast between matrix and lipid vesicles or collagen fibers. The effects of glycerol as observed in thin films explain some of the …

Aldehyde Fixation Causes Membrane Vesiculation During Platelet Exocytosis: A Freeze-Substitution Study, E. Morgenstern

Scanning Microscopy

Despite a plethora of reports on the ultrastructure of secretory granule release by exocytosis, the release of coagulant activity from stimulated platelets is still being attributed to membrane vesiculation. Membrane vesiculation and the formation of myelin figures have been shown to be artifacts of glutaraldehyde GA fixation. Cells fixed by direct osmium or rapid freezing are free of such structures. Yet there is still doubt that rapid freezing interferes with vesiculation process. This study has addressed this issue by examining: (1) whether freezing and freeze-substitution affects membrane vesiculation, (2) whether paraformaldehyde-fixation also induces the phenomenon, and (3) whether the aldehyde …

High Voltage Electron Microscopy And Low Voltage Scanning Electron Microscopy Of Human Neoplastic Cell Culture, Marek Malecki

Scanning Microscopy

Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias).

The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical …

Adsorption Staining Of Freeze-Substituted And Low Temperature Embedded Frog Skeletal Muscle With Cesium: A New Method For The Investigation Of Protein-Ion Interactions, L. Edelmann

Scanning Microscopy

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs⁺ -ions which are known to accumulate in living cells like K⁺ -ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be …