Comparison of Cryopreparation Techniques for Electron Probe Microanalysis of Cells as Exemplified by Human Erythrocytes

Erythrocytes in human blood were used to evaluate the reliability of cryopreparation techniques for electron probe X-ray microanalysis of biological cells and tissues. The elemental content determined by X-ray microanalysis of ultrathin freeze-dried cryosections was found to be consistent with data known from the literature. Considerable redistribution of the intracellular elemental composition was found after freeze-substitution as well as after freeze-drying followed by resin embedding. Two conclusions are drawn from this study: 1. Erythrocytes in human blood are a suitable reference specimen for evaluation of specimen preparation techniques for microanalysis. 2. At present, freeze-dried cryosections are the most reliable specimen type for quantitative electron probe microanalysis of cells

The Determination of Wet Weight Concentrations of Elements in Freeze-Dried Cryosections from Biological Cells

Energy dispersive X-ray microanalysis in STEM (scanning transmission electron microscope) of freeze-dried cryosections from biological cells provides information on the subcellular element distribution in terms of dry weight concentration. The local dry weight content in the range of 5-50%, respectively the local water content within 50 to 95%, in different subcellular compartments can be determined by measuring the darkfield intensity by means of an annular detector in STEM. Calibration is done by measuring the darkfield intensity of similarly prepared cryosections from dextran-water-solutions in varying concentration. Thus, by combining the X-ray microanalytical data evaluated by the continuum method with the STEM darkfield values, wet weight concentrations of elements in subcellular compartments are obtained. The method was applied to fibroblast cells in suspension. The reliability of this method is compared with other techniques to measure mass and intracellular water by electron microscope methods

Preparation of Cryosections for Biological Microanalysis

The element distribution in biological cells and tissues can be revealed by electron probe microanalysis from ultrathin cryosections. In particular, the distribution of physiologically important and often mobile elements such as Na, Mg, P, S, Cl, K, and Ca can be studied in cryosections on an ultrastructural level. The cryopreparation technique required for this purpose consists of 1. cryofixation, 2. cryosectioning, 3. cryotransfer including freeze-drying and carbon coating if necessary, 4. energy dispersive X-ray microanalysis in a cold stage equipped scanning transmission electron microscope. The lateral analytical resolution of this method is less than 50 nm in freeze-dried ultrathin (about 100 nm thick) cryosections. The detection limit is about 12 mMol/kg dry weight for elements with an atomic number higher than 12. For sodium this value is about 48, for magnesium about 36 mMol/kg dry weight. Good cryofixation without or at least with very small ice crystals with a diameter of 50 nm or less is found to be necessary not only for recognition of ultrastructural details but also for reliable evaluation of X-ray spectra. Carbon coating of frozen-hydrated sections reduces the mass loss observed in uncoated frozen specimens

Bild und Raum

Wissenschaftliches Kolloquium vom 19. bis 22. April 2007 in Weimar an der Bauhaus-Universität zum Thema: ‚Die Realität des Imaginären. Architektur und das digitale Bild

Morphological variation and genetic diversity of Triops cancriformis (Crustacea: Notostraca) and their potential for understanding the influence of postglacial distribution and habitat fragmentation

Triops cancriformis (Crustacea: Notostraca) occurs in ephemeral habitats like rain pools or floodplain pools distributed over a large geographical range. The named habitats are disturbed by human impacts and, consequently, T. cancriformis is endangered throughout its distribution range. In the present thesis the populated habitats and threats are characterised and further morphological and genetic variations detected among and within European populations are reported. On the basis of recent investigations it is shown that T. cancriformis subspecies separation is hampered by an individual variability which points to the necessity of species revision. The analysis of mitochondrial gene sequence data suggests that the species has colonised most of Europe very recently. The advantage of a complex reproductive strategy in T. cancriformis in this process is discussed. The population structure resolved with nuclear DNA markers highlights that there is low allelic diversity among and within populations compared to other Branchiopoda (Daphnia). By means of the present study it can be shown that habitat conservation is most important to protect T. cancriformis

Embryonic Development of the Pharyngeal Arch Arteries in Mammals

The pharyngeal arch arteries (PAAs) in mammals undergo asymmetric remodeling to give rise to the major blood vessels. The first and second PAAs form rudimentarily in mammalian embryos and eventually regress as the third, fourth, and sixth PAAs predominate. Cell migration, proliferation, and apoptosis drive the remodeling process, stimulated by a number of underlying molecular mechanisms. Sonic hedgehog, Hox genes, Tgfβ2, Tbx1, and a number of transcription regulators all influence PAA morphogenesis. Tbx1, which is found in the deleted region of Chromosome 22 in DiGeorge Syndrome patients, forms anterior-to-posterior and medial-tolateral gradients in the developing PAA system to promote remodeling. The transient and rudimentary presence of the first and second arteries may help establish the Tbx1 gradient, recruit cardiac neural crest cells, contribute to formation of the other arches, and advance craniofacial development

Preparation of Cultured and Isolated Cells for X-Ray Microanalysis

Various electron microscopical preparation techniques are compared with regard to the preservation of the intracellular element distribution as determined by X-ray microanalysis in scanning and scanning transmission electron microscopy. By use of chemical agents for fixation and dehydration ions are redistributed and washed out. This is also true for freeze-substitution. Whole cells are prepared by cryofixation followed by freeze-drying. Interference of intracellular measurements by extracellular elements can be avoided by appropriate washing the cells before cryofixation. The washing medium has to be carefully selected in order to avoid distortions of the original intracellular element content. These problems are circumvented by the preparation of freeze-dried cryosections from cryofixed cells. This is demonstrated by data of the intracellular elemental composition in cultured cells (fibroblasts, Staphylococcus aureus bacteria) and in cells isolated from rat tissue (kidney papillary collecting duct and liver). Cryofixation of a single cell in a defined functional state is illustrated by results obtained from streaming Amoeba proteus cells, cryofixed under light microscopical control. The main conclusion is that X-ray microanalysis of cells in functional states requires cryofixation and cryopreparation techniques which have to be adapted to the particular cell biological problem to be investigated

The Influence of Different Cryopreparations on the Distribution of Ions in Bullfrog Myocard Cells

Bullfrog heart muscle trabecula are shock-frozen in liquid propane cooled by liquid nitrogen and then processed for X-ray microanalysis in two different ways: 1. Freeze-drying followed by vacuum embedding. 2. Cryoultramicrotomy and freeze-drying. Stained sections of freeze-dried, embedded tissue exhibit detailed ultrastructure, but are useless for X-ray microanalysis. Unstained, dry cut plastic-sections are suitable for X-ray microanalysis, but the ultrastructure appears faint. Higher electron optical contrast and peak-to-background ratio of X-ray spectra are generally obtained in freeze-dried cryosections. Both preparation methods show that the X-ray spectra are influenced by the quality of cryofixation. The phosphorus/potassium ratio in nuclei increases with increasing ice crystal size