L. Jarczyk

The influence of target preparation and mode of irradiation on PIXE analysis of biological samples

Abstract The following methods of target preparation were examined and compared: dry ashing at high temperature, low temperature ashing in plasma asher, wet ashing, lyophilization at a temperature of 35°C, cryofixation with drying in vacuum and dehydration in alcohol with drying in vacuum. All these techniques were applied to prepare targets from five different rat organs: liver, kidney, brain, lung and muscle tissue. The dried and powdered sample material was pressed into pellets or was distributed on formvar film. The evaporation of the thin carbon layer on the investigated target and placing of the thin carbon film in front of a target were also tested. The targets were irradiated in vacuum using an external beam in the air chamber. The influence of the method of target preparation on the detection limits, time requirements and escape of elements from the sample material is discussed.

Proton microbeam study of calcium-phosphate complexes in human arteries

Abstract The proton microprobe was used for the determination of the elemental composition of Ca-P deposits in 3 types of human arteries (abdominal aorta, coronary artery, circle of Willis). The techniques used for the elemental analyses were PIXE and RBS. The distribution of concentrations of C, N, O, P, S, Cl, K, Ca, Fe, Cu, Zn, Br, Sr and Pb were measured. The chemical structure of the Ca-P deposits as well as the role of certain elements in the calcification process were considered. A comparison between different investigated arteries was also made.

The analysis of mineral deposits and proteoglycans content in the cartilage of mouse trachea using PIXE in combination with proton microprobe

The tracheal cartilage of mature mice have been investigated using PIXE (proton induced X-ray emission) in combination with a proton microprobe on snap frozen cryosectioned material. The localization and quantitative measurements of P, S, Cl, K, Ca, Fe, and Zn concentrations as well as direct and indirect assessment of glycosaminoglycans by measurement of S content and measurement of bound colloidal iron at pH = 1.8 has been performed. Adjacent sections were stained with the Hale method in Müller modifications and the v. Koss method for sulphated mucins and inorganic deposits respectively. It has been found that hyaline cartilage in trachea contains mineral deposits and that P + Ca amounts up to 22% of cartilage dry mass. The Ca/P ratio approaches 2 what indicates hydroxyapatite type crystals. The cartilage contains substantial amounts of S reflecting the presence of sulphate groups. It was found that the cartilage binds also colloidal iron at low pH. There is a good correlation between places with high amount of bound colloidal iron assessed by PIXE and places showing strong Prussian Blue staining. The Fe/S ratio was, however, much lower in the cartilage than in other tissues what indicates that the colloidal iron method does not give quantitative results. There were no regions showing substantial decrease in Fe/S ratio which we found previously as typical for degenerating and calcifying growth plate cartilage. This may be connected with a relatively low degree of calcification degree of the tracheal cartilage.

Experimental comparison of micro-PIXE with other methods utilized for biomineralization studies

Abstract The present study deals with the investigation of arterial wall mineralization i.e. of inorganic compound development within artery wall under normal or pathologic conditions. Autopsy samples of human aorta as well as fragments of aorta obtained from hypercholesterolemic rabbits were used for the experiments. The samples were investigated using micro-PIXE, PIXE and PIGE techniques, infrared and Raman spectroscopy, X-ray powder diffraction and a variety of histochemical methods in order to compare the techniques based on a proton microprobe with the other methods which aimed at the investigation of inorganic deposits. Proton microprobe measurements confirm the different composition of mineral deposits found in rabbit and human aorta samples. In the case of animal aorta the apatite crystals constitute the mineral form of deposits while for human samples we deal with a mixture of different compounds. Moreover, for rabbit aorta samples the deposits were found always in the atheromas, while in human aorta sections the mineral deposits were localized in the media of the aortic wall. The proton microprobe can be considered to be a valuable addition to the experimental methods that have been applied in the study of the biomineralization process. It permits a qualitative determination of mineral deposits in situ while infrared and Raman spectroscopy as well as X-ray powder diffraction measurements may be performed only for ashed aorta samples. The possibility of in situ measurements and high detection sensitivity seem to be the most significant advantages of a proton microprobe in the study of artery wall mineralization.

Determination of circulating blood volume using PIXE

Abstract The determination of the circulating blood volume using the PIXE method for measuring the time variation of the tracer concentration is described. The stable atomic tracer contained in widely-used drugs is introduced into the vascular system by injection. Two drugs containing bromine and iodine as tracers were tested. The experiment was performed in rabbits and in man. Additional information could be extracted from the shapes of the time variation of the concentration curve.

Micro-PIXE quantification of histochemical reactions

Abstract The proton induced X-ray emission in combination with a proton microprobe (micro-PIXE) was used for the quantitative determination of a locally bound histochemical reaction end-product. The determination of proteoglycans (PG) content in the cartilage is described in detail. The cartilage from the femur and the skull of 17–19 days old embryos and from ribs of mature CBA strain mice was investigated. The 8 μm cryomicrotome sections, placed on a formvar backing, were incubated in colloidal iron or in alcian blue (dye molecule contains Cu) solutions. The determination of PG contents was made indirectly by assessment of the Fe or Cu concentrations. Differences in the PG contents were observed within all investigated cartilage types. The embryonal cartilage contains more PG than that obtained from mature animals. For the cartilage from the femur the highest content of PG was found in the mineralization front while fully formed bone trabeculae exhibited a PG level 10 times lower. In the case of intramembranaceous ossification the content of PG was also higher in the skull cartilage than in the loose connective tissue. On the basis of the performed studies some methodological conclusions can be drawn. For PG determination, the alcian blue method is more reliable than the colloidal iron technique. The review of the histochemical procedures which may be combined with the micro-PIXE method is also given.

Comparison of global elemental composition of liver tissue with composition of different cell fractions

Abstract The distribution of each element obtained by micro-PIXE analysis should be verified by correlating it with the tissue microscopic structure. In order to facilitate this we tried to correlate the measured distribution of various elements with the elemental composition of different cell fractions. For this purpose the various rabbit liver fractions were separated and analysed by PIXE using a broad proton beam.

Proton microprobe studies of the mineralization process in selected organic matrices

Abstract The application of the proton-induced X-ray emission method in combination with a proton microprobe (micro-PIXE) to studies of the mineralization process in selected organic matrices is presented. The flat-bone mineralization of the skull of 17–19 days pregnancy mouse embryos is described in detail. The section thickness as well as concentrations of P, S, Cl, K, Ca, Zn and Br were determined using the raster- or line-scan irradiation mode. On the basis of Ca and P contents the amount of inorganic phase was determined for each sample under investigation, while the Ca/P ratio was used for the identification of the chemical compounds within inorganic material. In the embryonal bones we observed almost stoichiometric hydroxyapatite; however, in the intramembranaceous ossification of the young (17 day) animals, octacalcium phosphate was identified. At the given age of the embryo the amount of minerals is much higher in the femur bone than in flat bone. The micro-PIXE method furthermore turned out to be sensitive enough to study the mineralization accompanying malignant tumors. We have also observed the involvement of some trace elements in the development of the mineralization process. The last two observations are unattainable by histological staining.

A PIXE method for investigating the time changes of halothane content during anaesthesia

Abstract The PIXE method was applied to determine the time variation of the bromine content in blood during and after anaesthesia. A mathematical model to describe the processes responsible for the bromine distribution is proposed.