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

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

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

Elemental Mapping of Cryosections from Cnidarian Nematocytes

The distribution of elements in stinging capsules containing cells called nematocytes is shown by pseudocoloured maps representing the X-ray intensity collected from freeze-dried cryosections. This method provides a distinct overview in addition to the quantitative evaluation of single X-ray spectra. Selected examples illustrate the elemental compartmentation in various cnidarian animals. In particular the matrix of capsules in Hydra vulgaris contains high amounts of K in comparison to the tubule, the surrounding capsule wall and the cytoplasm, whereas in Actinia eguina capsules have either high concentration of Ca or Mg, the latter accompanied by S

Electron Probe X-Ray Microanalysis of Epithelial Cells: Aspects of Cryofixation

Content and distribution of diffusible ions in epithelial cells were studied by scanning transmission electron microscopy and energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections from trout kidney, rat liver and Malpighian tubules of Drosophila larvae. Cryofixation of small excised kidney and liver samples by rapid immersion into liquid propane resulted in intracellular K/Na-ratios \u3c 1. In contrast, K/Na-ratios \u3e 7 were obtained after in situ cryofixation by means of a cryopunching device which allows tissue pieces to be frozen during excision from the intact organ. Isolated hepatocytes cryofixed in a small droplet of culture medium had a K/Na-ratio of 3.7. After culturing the hepatocytes, the K/Na-ratio increased to 24. Effects of extracellular media of different composition on the intracellular element content were studied. Malpighian tubules of Drosophila larvae were cryofixed by rapid immersion into liquid propane, and the distribution of K across the cells forming the tubules from the basal to the apical cell membrane was measured. An increasing K gradient was found from the intermediate to the apical cytoplasm. The intracellular K distribution was dependent on ions and transport inhibitors present in the fluid surrounding the Malpighian tubules within the larvae. Content and distribution of ions in epithelial cells sensitively depend on the physiological state immediately before cryofixation. Thus, electron probe X-ray microanalysis of cells and cell functions requires careful selection and control of the cell system to be studied

Analysis of Early Hard Tissue Formation in Dentine by Energy Dispersive X-Ray Microanalysis and Energy-Filtering Transmission Electron Microscopy

Thin cryosections and sections of embedded tissue were prepared from dentine of cryofixed rat incisors. Energy dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have been applied to study the calcium and phosphorus distribution in predentine of these incisors. A small enrichment of calcium and phosphorus was found in the predentine zone near the dentine border. Element distributions were correlated with analyses of the early crystal formation in dentine. These investigations were carried out by parallel applications of electron spectroscopic diffraction (ESD) and electron spectroscopic imaging (ESI) using zero-loss filtering. It was found that the earliest crystal formations already showed the lattice of the hexagonal mineral apatite. They form parallelly arranged chains of dots which coalesce rapidly to form needle-like crystallites along the collagen microfibrils

Limitations and Prospects of biological electron probe X-ray microanalysis

Analytical electron microscopy is used to identify, localize and quantify chemical elements in biological samples with the aim to correlate the element distribution in cells and tissues with the particular functional state. Energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections is considered to be the most efficient technique with respect to spatial analytical resolution and detection limit. The potentialities and limitations of this technique are illustrated by results from 3 current research projects: Element compartmentation in hepatocytes, cytotoxicity of an organotin compound, and calcium movements during osmoregulation of epithelia. At present, the absolute detection limit is 170 atoms in a minimum analytical volume of 30 x 30 x 100 nm(3). X- ray microanalysis of cryosections from biological samples is limited by ice crystal growth during cryofixation and by the poor X-ray collection efficiency in most electron microscopes. Therefore, the construction of an analytical electron microscope dedicated to biological X-ray microanalysis is proposed. This microscope features considerably improved X-ray collection efficiency due to an annular X-ray detector. The absolute detection limit is estimated to be less than 10 atoms in a minimal analytical volume of 10 x 10 x 100 mn(3) of a cryosecti