Transmission electron microscopy and scanning probe microscopy.
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Animals↗
Biomedical subjects
K L Klomparens
Publications and source records attributed to K L Klomparens.
Characterization of agarose as an encapsulation medium for particulate specimens for transmission electron microscopy.
Agarose, agar, and gelatin were initially compared as encapsulation media for 3 structurally diverse particulate specimens: bacteria, yeast, and mitochondria. Agarose proved superior to both gelatin and agar for ease of handling and overall image quality (minimum background). All sample types exhibited high quality fixation and structural detail with no heat damage from the agarose medium. Based on this finding, we further characterized agarose encapsulation as affected by post-fixation, en bloc staining and resin type. Osmium tetroxide post-fixation, followed by en bloc uranyl acetate staining, could be performed without an increase in the electron density of the encapsulation medium. Agarose proved successful as an encapsulation medium regardless of the resin type or preparation protocol, thus providing flexibility in experimental design and excellent results over a range of variables.
Animals↗
Comparison of bromine and permanganate as ultrastructural stains for lignin in plants infected by the fungus Colletotrichum lagenarium.
Transmission electron microscopy (TEM) and energy dispersive X-ray microanalysis (EDS) were used to localize manganese from KMnO4, and bromine, as ultrastructural stains for lignin in an herbaceous plant. The Spookie cultivar of pumpkin is susceptible to infection by the fungus Colletotrichum lagenarium and served as a model system to compare the Br and KMnO4 techniques. Bromine was used in a fixation/staining procedure, and in separate experiments, KMnO4 was used as either a fixative or as a postsection stain. The technique for using bromine was modified from the woody plant procedure by adding a paraformaldehyde prefixation step. With the bromine procedure, cell walls were well-preserved, but the cytoplasm was heavily extracted. The KMnO4 procedures produced well-fixed cytoplasm, but with some staining artifacts. With all procedures, EDS dot mapping demonstrated lignin deposition in the cell walls specifically associated with sites of fungal infection. Lignin was also localized in secondary walls of tracheary elements, sites known to be highly lignified. The bromine procedure provided the most specific localization of lignin with a minimum of artifact. The specific applications of these stains provided data on the ultrastructural localization of lignin which contributed to the elucidation of its role in the interactions between pathogenic fungi in both their resistant and susceptible plant hosts.
Bromine↗
Membrane properties of a plant-pathogenic mycoplasmalike organism.
In terms of biosystematics, the plant-pathogenic mycoplasmalike organisms (MLOs) have been tentatively placed into the class Mollicutes. Certain physiological tests have been used to distinguish families within this class: the sterol-nonrequiring Acholeplasmataceae differ from the sterol-requiring Mycoplasmataceae in that the former are more resistant to lysis by digitonin and more sensitive to lysis in hypotonic salt solutions. To test MLOs for these membrane properties and thus assist in their definitive classification, a dot-blot microassay procedure was used to detect nucleic acids released from lysed cells. The results show that MLOs resemble acholeplasmas grown in the absence of sterols in that they are resistant to digitonin and sensitive to hypotonic salt solutions. The MLOs can be differentiated from acholeplasmas grown without sterols by their greater resistance to lysis in hypotonic sucrose solutions.
Acholeplasma laidlawii↗
Effects of substrate texture and curvature on the morphology of cultured cells.
The ultrastructural characteristics of several growth matrices were examined using two cell types chosen for their distinct growth habits. Chinese hamster ovary cells and Balb-c 3T3 mouse fibroblasts were grown on flat substrates (glass, tissue culture plastic, Millipore filters) as well as spherical (glass, tissue culture plastic, cross-linked dextran) substrates. Cells were plated maintaining equal densities and growth surface area. Once the majority of the cells reached confluency, the cell's morphology on each matrix was examined using scanning electron microscopy. Digital analysis was performed on cell attachment area to compare the effect of each matrix on cell spreading. Variation in cell shape was dramatic between matrices, being most noticeable between a textured surface (filter, dextran bead) and that of a smooth (glass) surface. Even within smooth surfaces, some variation was observed. There was also an effect of matrix curvature on cell attachment area, the greatest being in the 3T3-c Balb cells, causing an overall decrease in the area of attachment between cell and matrix. The changes seen could also be related to the particular cell type used. Hamster ovary cells tended to be cylindrical and showed little effect between matrices, whereas the mouse fibroblasts, which are more flattened, showed the matrix effect to a greater degree. This study demonstrates the necessity of being aware of substrate-induced cell changes in tissue culture, where some variation in cell shape may be due to the surface on which the cells are grown as opposed to the experimental procedure.
Analog-Digital Conversion↗
The development and application of ultrastructural research in mycology.
Electron microscopy has contributed a great deal to the field of mycology. Fungal ultrastructure has been, and continues to be, a key research element in the study of spore development and germination, host-pathogen interactions, nuclear behavior, and studies of subcellular organelles and organization linking structure and function. Since the earliest research in transmission electron microscopy in the 1950s, mycologists have kept pace with the developments in all areas of electron microscopy and have used them to great advantage in generating fine structural information on fungi. These recent developments include the use of scanning electron microscopy in the 1960s, X-ray microanalysis, cryopreservation and immunoelectron microscopy in the 1970s and 1980s. All of these techniques will continue to provide mycologists with the means to gain morphological and analytical data at the ultrastructural level.
Cryopreservation↗
Direct Comparison of Phosphate Uptake by Adnate and Loosely Attached Microalgae within an Intact Biofilm Matrix.
We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte Potamogeton illinoensis or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosely attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source.
Journal Article↗
Gold labeling of cell monolayers grown on dextran beads.
Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.
Animals↗