Scanning microscopy. Supplement最新文献

In cell biology, electron probe X-ray microanalysis can reveal the distribution of chemical elements inside a single cell. The full description of a biological system (cell population, tissue) requires a great number of spot measurements. In quantitative analysis, the measurements are subject to experimental errors of several types; moreover, the relations between the resulting values are usually more interesting than the absolute concentrations. Nevertheless, the proper evaluation of quantitative values can discover information more on the object of study. A system of simple statistical tests is suggested here which can solve several problems. Some concentration values can be far from the statistical average due to errors in measurement; therefore, a statistical test of plausibility of the measured values is carried out. In the compartments (e.g., nucleus, cytoplasm or other selected areas), the distribution of an element can be nonhomogeneous, and hence a statistical test of homogeneity of the element distribution in specified areas is provided. The tests continue with a test for correlation, in which the concentrations of a given element in a pair of specified areas are compared. These test proceed step-by-step for all elements of interest. Subsequently, the relations of concentrations in all possible pairs of elements in the area in question are calculated. Moreover, cells within a population can be different from the point of view of elemental concentration; a statistical test of homogeneity of the cell population is provided. In the case of nonhomogeneity, the concentration values and/or cells within a population are clustered into homogeneous groups. The evaluation is carried out automatically, with a simple program. The system of programs, in which the program for evaluation is incorporated, is included semi-on-line in the EDAX9900 system, where the measurement and evaluation are carried out in sequence. The results for a population of Streptomyces aureofaciens are shown as an example.

X-ray microanalysis of non-biological and biological specimens was carried out in an environmental scanning electron microscope (ESEM) over a range of atmospheric conditions. Introduction of water vapour into the specimen chamber lead to direct X-ray contribution from oxygen atoms, an increase in extraneous background (causing reduced P/B ratios of other elements), X-ray absorption (also reducing P/B ratios) and broadening (skirting) of the electron beam. Similar results were obtained after introduction of an argon atmosphere. These effects were reduced under conditions of minimal chamber atmospheric pressure and maximal accelerating voltage. Because of beam skirting, quantitative X-ray microanalysis of biological specimens in a water vapour atmosphere was only valid where the sample was spread over a wide area (leading to mean elemental values for the whole preparation). Unless appropriate correction factors or changes in instrumentation can be implemented, quantitative analysis of wet specimens in ESEM cannot be applied to discrete specimens or to limited areas within a mixed sample.

For biological X-ray microanalysis, cryoembedding (CE) combined with cryofixation (CF) and cryodehydration (CD) was already proposed as an alternative method to freeze-dried cryosections in 1984 by Wróblewski and Wroblewski. CD by freeze-drying (FD) is usually recommended because it provides better retention of diffusible elements. CD by freeze-substitution (FS) has the advantage of being simpler, giving more reproducible preservation of ultrastructure and causing fewer problems for resin infiltration. We have increased the retention of diffusible elements by using home-made devices for CS and CE in the new Lowicryl K11M and HM23 resins. These resins allow samples to be kept at a maximum temperature of 213K and 193K respectively. Application of multivariate statistical analysis (MSA) to X-ray data (spectra and maps) allows the study of correlations between the analyzed elements in different nuclear areas and in the cytoplasm. The "factorial" images, obtained with MSA, display the compartments of strong correlation between P and K (nucleic acids) and the compartments of strong correlation between S and K (proteins). We suggest that the future application of MSA methods will provide increased knowledge of the physio-pathological compartmentation of diffusible elements at the subcellular level.

The availability of a cryotransfer stage, highly efficient electron energy loss spectrometers, and ultra-thin-window energy-dispersive x-ray spectrometers for the VG Microscopes HB501 field-emission scanning transmission electron microscope (STEM) provides this instrument with the potential for high resolution biological microanalysis. Recent technical advances offer cryosections that are thin enough to take advantage of the analytical capabilities of this microscope. This paper first discusses the quantitative characterization of freeze-dried, ultrathin cryosections of directly frozen liver and brain by low-dose dark-field STEM imaging. Such images reveal high-quality sections with good structural detail, mainly due to reduced preparation artifacts and electron beam damage. These sections are thin enough for dark-field mass analysis, so that the mass of individual organelles can be measured in situ, and their water content deduced. This permits the measurement of mass loss-corrected subcellular elemental concentrations. The results suggest several new applications for cryosections as illustrated by data on synaptic activity-dependent calcium regulation in Purkinje cells of mouse cerebellum. Low-dose mass analysis of cryosections in combination with x-ray and electron spectroscopy is a promising approach to quantitating physiological changes in mass distribution and elemental composition.

The X-ray microanalysis of thin biological samples which are usually supported on a thin organic film or are self-supporting specimens, has required the use of standards which contain the elements of interest. Spectra from the standards are used to calculate the factors for converting X-ray data recorded on the specimen into elemental concentrations. A method is discussed here, in which these factors are evaluated from formulae. The most important physical process to be evaluated is that of characteristic X-ray production in the specimen. The bremsstrahlung production must also be evaluated if the Hall or continuum normalisation (CN) method of quantitation is to be used. This paper discusses briefly methods of calculating values for the X-ray production cross-sections for both characteristic and bremsstrahlung radiation. The way in which these are incorporated into standardless quantitation methods for biological samples is described. Calculations of some cross-section data are presented for typical analytical conditions.

X-ray microanalysis was used to study elemental distribution in Malpighian tubule cells of Locusta migratoria and how these are affected by the replacement of bathing medium K+ with Rb+ and by inclusion of the transport inhibitors ouabain and n-ethyl maleimide (NEM) in standard (K+-containing) and Rb+-Ringer (K+-free) solutions. Incubation of tubules in standard Ringer containing 1mM ouabain dramatically affected the intracellular levels of K and Na. The intracellular K concentration fell and Na concentration increased in all regions studied. Despite this, a gradient of increasing K concentration from basal to apical cell surface was maintained. Ouabain also reduced the intracellular levels of Rb when applied in Rb+-Ringer. Cl and P levels were unaffected by ouabain treatment. Incubation in standard and Rb+-Ringer solutions containing 1 microM NEM caused a significant increase in intracellular K levels in all regions of the cell compared with that observed in the absence of NEM. Rb levels were little affected by NEM except in the apical cytoplasm and microvillar regions where they were significantly reduced compared with Rb+-Ringer controls. NEM effected a significant increase in cellular levels of Na under Rb+-Ringer conditions. Intracellular Cl and P were not significantly affected by NEM. These results are discussed in relation to proposed mechanisms for the transport of ions and water across this secretory epithelium, with particular emphasis on the role of K+ as the 'prime mover' in this process.

An overview is given of laser microprobe mass spectrometry (LMMS) in biology and biomedicine (1989-1993). The present instrumentation and its analytical features are surveyed. Applications are presented with special attention on human and animal tissue samples, as well as plant material. The capabilities of LMMS to study the element distribution in histological sections, to identify the chemical composition of inorganic inclusions and to generate structural information from organic compounds are evidenced.

Elemental redistribution induced by insulin secretion, was investigated by electron and proton probe X-ray microanalysis. In particular, ion fluxes following immediately upon stimulation were studied. As the sensitivity of the electron probe was insufficient, the proton microprobe was employed. In order to see whether the cell is asymmetric with respect to Ca2+ influx, the cells were stimulated in the presence of Sr2+ (as a Ca2+ analog). Insulin-secreting cells (RINm5F cells and isolated mouse beta-cells) were cultured on grids and shock-frozen at 2-30 seconds after stimulation. In a large number of cells, the major elements and and large fluxes were analyzed by the electron microprobe. In the proton microprobe, selected cells were analyzed and elemental maps were compared with electron micrographs of the same cells. The proton microprobe, but not the electron microprobe, could detect an influx of Sr in response to K+-stimulation for 2 seconds, in RINm5F cells. No polarization of Sr2+ uptake in RINm5F-cells could be detected, and the beta-cells did not respond to high K+ by uptake of Sr. Momentary stimulation of beta-cells also resulted in a significant increase in Na, detected by the electron probe. Spreading of the beta-cells on the substrate appears to influence the subcellular elemental distribution. Thus, the proton probe has potential to detect small changes in elements such as those occurring after short-time stimulation.

The use of X-ray microanalysis in human pathology may require the use of cryoprepared tissue. Often it is impossible to carry out freezing of the tissue in an optimal way, and in addition, it is difficult to carry out experiments in living patients. The use of in vitro systems and cell cultures allows separation of the process of tissue removal and the freezing procedure, and also makes testing of pharmacological or toxic substances possible. In experiments with animal tissue it was shown that incubation in a physiological buffer induced significant changes in the concentrations of Na, K, and Cl. In general, the concentrations of Na and Cl increased, those of K decreased. Prolonged incubation of brain tissue (cortex and hippocampus) and of liver resulted in further changes of the cellular ion contents in the same direction. Incubation of pancreas and submandibular gland resulted in a limited reversal of the changes induced by dissection. The submandibular gland in vitro showed the same response to cholinergic stimulation as the gland in situ. The use of cell cultures for X-ray microanalysis is briefly reviewed and illustrated by an example of analysis of an immortalized sweat gland cell line. It was shown that these cells respond to stimulation by cAMP with loss of Cl and that this response was unaffected by the type of substrate the cells were grown on.