[en] An X-ray microanalytical and morphological investigation has been carried out on rapidly frozen, freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerization procedures following freeze-substitution and freeze-drying. The investigation also included an analysis of specimens infiltrated, embedded and polymerized by ultraviolet irradiation at low temperatures with Lowicryl HM20. The morphological preservation of Lowicryl embedded tissue was adequate for the identification of different cell structures like nuclei, mitochondria, lysosomes and different types of endoplasmic reticulum. X-ray microanalytical investigation of low temperature embedded material displayed an elemental composition of cells and organelles similar to that found in freeze-dried cyosections. Compared with freeze-dried cryosections, low temperature embedded material could be sectioned for light microscopy and area of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogenous cell populations

[en] The elemental content of differentiating chondrocytes in situ and in vitro was studied by X-ray microanalysis. The in situ studies were carried out on semi-thick cryosections and on thin sections of freeze-dried low-temperature vacuum-embedded rat rib growth plate. Resting, proliferative, and hypertrophic cells were analyzed separately. The in vitro studies were carried out on cultured cell fractions isolated from rat rib growth plate. The cell fractions were relatively homogeneous and contained resting - early proliferative, proliferative or hypertrophic cells, respectively. Data from cells in vitro are generally not comparable to data obtained from cells in situ. Chondrocytes in situ contain relatively high levels of Na, which is probably due to: (1) high Na levels in the cartilage matrix formed intracellularly, and (2) low supply of oxygen and nutrients. High Na levels in (some of) the proliferative cells may also be related to mitotic activity. The chondrocyte matrix contains relatively high levels of K. The data in the present study are compared to those of previous studies on the elemental content of chondrocytes

[en] X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

[en] Although X-ray microanalysis in the electron microscope is the most common method for microanalysis of biological specimens, other methods of elemental microanalysis (electron energy loss spectroscopy, scanning Auger microanalysis, and proton, ion, and laser microprobe analysis) may provide important complementary information and help overcome some of the limitations of electron probe X-ray microanalysis. Despite differences in physical principles and instrumentation, the various microanalytical methods have much in common with regard to specimen preparation, quantitative analysis, and interpretation of analytical data. A common approach to microanalytical problems in the biological sciences, irrespective of the analytical techniques used, seems therefore indicated

[en] Many diseases are associated with a change in the distribution of diffusible ions at the cell or tissue level. These diseases can profitably be studied by X-ray microanalysis. This technique for the study of ion distribution requires the use of cryoprepared specimens. Analysis at low or medium resolution can be carried out on thick or semi-thick cryosections, or on frozen-hydrated or freeze-dried embedded bulk samples. Such analyses are particularly useful in the initial stages of an investigation or when data from a large number of samples have to be acquired. Also X-ray microanalysis of cultured or single cells prepared by freeze-drying can be used to rapidly collect information on a large number of cells. Analysis at high resolution has to be carried out on thin sections: Cryosections or sections of freeze-substituted or freeze-dried embedded tissue. For the latter type of specimens, the use of low-temperature embedding methods may have important advantages

[en] Possible alternatives to rapid freezing in liquid nitrogen of tissue for X-ray microanalysis of electrolytes at the cellular level were investigated. These alternatives might be used in cases where tissue becomes available for examination, e.g., at autopsy, but liquid nitrogen is not immediately available. Rat submandibular gland was used as a test tissue. Freezing of pieces of tissue in a conventional freezer at -80 degrees C or even at -20 degrees C retained the elemental distribution at the cellular level, and also retained the difference between a normal and a pathological situation. Storage of tissue in a refrigerator, or delaying the autopsy in anticipation of the arrival of liquid nitrogen is not recommended. Significant changes in the cellular ion content occurred if the tissue was left in the animal for 24h post-mortem

[en] Since energy dispersive X-ray microanalysis cannot distinguish between isotopes of the same element, alternative methods have to be used to get information similar to that obtained in experiments with radioactive tracers. In the present study, strontium was used as a tracer for calcium, and bromide as a tracer for chloride. Rats were injected with strontium chloride in vivo, and the uptake of strontium in the acinar cells of the submandibular gland was studied. Eventually a Sr/Ca ratio of 0.3 was reached. In some animals, secretion of mucus had been elicited by stimulation with isoproterenol 4 h prior to injection of strontium chloride. Exchange of calcium for strontium was enhanced by prior injection with isoproterenol. In a second experiment, rats were injected with sodium bromide, and the uptake of bromide by the submandibular acinar cells was followed in time, both in pilocarpine-stimulated and unstimulated glands. Under the experimental conditions, bromide was rapidly taken up by the cells, and the cellular Br/Cl ratio was close to that found in serum. Submandibular glands take up Br somewhat faster than other tissues (liver, heart muscle, skeletal muscle). The uptake of Br in pancreatic acinar cells was studied in vitro. These experiments showed a 1:1 ratio (molar) exchange of Cl for Br

[en] The validity of freeze-drying and low temperature embedding in Lowicryl resins has been investigated in studies of ion distribution in mouse embryological inner ear, rat rib growth plate, liver and submandibular gland. The morphological preservation of the tissues was adequate for the identification of different intracellular compartments and extracellular structures. It was also possible to analyze extracellular fluids in the vestibular part of the developing inner ear. Compared with thin cryosections, Lowicryl sections are easier to produce and are more stable during analysis. Freeze-dried embedded material can be easily reorientated during cutting and adjacent sections can be used for other purposes such as histochemical and morphological investigations. We found that hydrophilic Lowicryl K11M, which is normally used for immunocytochemical investigations, also can be used for microanalysis. That opens the possibility for combined immunocytochemical and microanalytical studies. However, infiltration and polymerization steps have to be carried out at slightly higher temperatures than when the hydrophobic Lowicryl HM23 is used

[en] X-ray microanalysis, neutron activation analysis and atomic absorption spectrometry were performed on normal and injured skeletal muscle. X-ray microanalysis of tenotomized rat soleus muscle showed significantly elevated levels of sodium and chlorine and lower potassium compared with normal muscle. Similar ion shifts could be demonstrated by neutron activation analysis and atomic absorption spectrometry. The concentrations of sodium and chlorine obtained by these techniques were somewhat higher and that of potassium lower than the values obtained by X-ray microanalysis. This can probably be attributed to the fact that in atomic absorption spectrometry and in neutron activation analysis the entire muscle biopsy contents are measured while in X-ray microanalysis only the content of muscle cells unaffected by extracellular, non-muscular components are determined. It can be concluded that X-ray microanalysis is a reliable technique to study the elemental content of biological tissue, especially tissue undergoing pathological changes affecting the extracellular spaces. Other types of analysis should be used when elements not detectable by X-ray microanalysis are of interest

[en] The site of attack of OH radicals on dihydrouracil and five methylated derivatives was determined by pulse radiolysis using N,N,N',N'-tetramethylphenylenediamine (TMPD) to detect oxidizing radicals and tetranitromethane (TNM) as well as K₃Fe(CN)₆ to detect reducing radicals. In dihydrouracil OH radicals abstract preferentially an H atom at C(6) giving the 6-yl radical (>= 90 %) which at pH equivalent to 6.5 reduces TNM and K₃Fe(CN)₆ at almost diffusion-controlled rates. Only a small fraction of OH radicals abstract the H atom at C(5) (<= 10%). The resulting 5-yl radical oxidizes TMPD to TMPD⁺ at pH 7-8. With the methylated derivatives of dihydrouracil, OH radicals react less selectively, especially in the case of N(1)-methyl derivatives. This methyl group is activated to a similar degree as the methylene group at C(6). In 1-Medihydrouracil the yield of N(1)-CH₂ radicals is about 29%. Radicals at the other methyl substituents are generated to a lesser extent (<= 10%) and are relatively unreactive towards oxidizing agents such as TNM and K₃Fe(CN)₆ as well as towards the reducing agent, TMPD. Although methyl substitution opens new routes for OH attack the preferred site of H abstraction remains C(6) (> 60%). (author)