The Hall Method in the Quantitative X-Ray Microanalysis of Biological Specimens: A Review

In the two decades since its inception by T.A. Hall, the continuum theory of quantification has become the general method for quantitative analysis of biological specimens. Although the method was originally developed for thin specimens, its use has been extended to thicker specimens, and it has also been used in quantitative determinations of local water content. The single most important difficulty in the application of the Hall method is the accurate calculation of the extraneous continuum, i.e., the continuum due to non-specimen sources. The different variations in methods for quantitative analysis of local water content are critically compared and a generally applicable method is proposed

Cryopreparation of Tissue for Clinical Applications of X-Ray Microanalysis

A number of diseases is associated with changes in ion and/or water distribution at the tissue or cell level, and X-ray microanalysis can be used to investigate the relationship between the disease process and the changes in elemental distribution. For analysis of diffusible elements by X-ray microanalysis, the tissue has to be prepared by cryotechniques. To carry out studies of this kind in a clinical environment poses a number of problems. Some of these problems occur already before the tissue is frozen, others are caused by the practical and ethical limitations that are imposed on the freezing method itself when human tissue is to be used. The use of cryostat sections for analysis at the cellular level, and of in vitro systems and cell cultures in which sampling and cryopreparation can be separated in time and place can be useful alternatives

The Correction for Extraneous Background in Quantitative X-Ray Microanalysis of Biological Thin Sections: Some Practical Aspects

The correction for extraneous continuum is of great importance in the quantitative analysis of thin sections of biological tissue. Although a theoretical model for this correction is available, its application in practice meets with problems. In this paper, a model system, consisting of sections of homogeneous plastic on copper mesh grids was used to identify sources of inaccuracies in the quantitative procedures. An unmodified electron microscope was operated under standard analytical conditions. It appeared that geometrical factors connected with the position of the analysis relative to the grid bars were of prime importance. The correction for the contribution of the support film to the continuum should ideally be carried out at the same location with respect to the grid bars as the matching measurement on the section. Also the position of the analysis with respect to its coordinates on the grids is important, in particular when the possibility of absorption of X-rays by the specimen holder exists. The use of slot grids (rather than mesh grids) may alleviate this problem at least in part. Additional factors of importance are differential mass loss in specimen and film, as well as undetected variations in specimen current

Calcium and Cystic Fibrosis

Cystic fibrosis (CF) is a generally lethal, congenital, genetic disease of unknown etiology. It is likely that a defective regulation of ion and water transport in exocrine glands and possibly also in other epithelial cells has a central role in the pathogenesis of this disease. Calcium has been implicated in the basic defect underlying CF because of findings of abnormally high calcium levels in some secreted fluids and some cells of CF patients. Using X-ray microanalysis, we have demonstrated elevated calcium concentrations in cultured fibroblasts and in goblet cells of the bronchial epithelium of CF patients. A factor produced by CF fibroblasts in culture can increase the calcium concentration in healthy cells, although this may be an indirect effect. In animal models for CF, such as the chronically reserpinized rat and the chronically isoprotere-nol-treated rat, abnormally high calcium levels in the acinar cells of the submandibular gland could be demonstrated, similar to the situation in CF patients. In the acinar cells of the parotid gland in these animal models, the calcium levels are, however, abnormally low. This suggests that the changes in cell calcium content are secondary to other changes, possibly changes in the secretory proteins. A study of the effect of the serum calcium level and of the calciotropic hormone calcitonin suggested that neither of these factors could be directly linked with CF. It is concluded that several lines of evidence point to a secondary rather than a primary role for calcium in the pathogenesis of CF

Simple cryogenic infrared window

A simple, cheap technique is reported that allows materials with both large and small thermal expansion coefficients to be mounted as windows in low temperature cryostats while at the same time avoiding thermal stresses. The construction may be thermally cycled many times with no change in its properties. It can hold differential pressures of at least 1 atm for a 1‐in.‐diam window. This technique seems to be particularly useful for applications where the use of soft materials cannot be avoided (e.g., ZnSe in the infrared)

Resonance‐enhanced low‐pressure optoacoustic cell

A low‐pressure optoacousticcell is described that can be used to lock the emission frequency of a laser. A model is developed which describes the low‐pressure behavior of the optoacousticcell as a function of cell dimensions, gas properties, and operating pressure.Resonantoptoacousticcells are predicted to improve the acoustic signal levels significantly. Experiments were carried out with a cell filled with CF4. The model was found to accurately predict resonator quality, resonance frequency, and acoustic response for pressures ranging from 0.1 to 3.0 kPa. At these low pressures acoustic attenuation processes, slow vibration to translation (VT) relaxation and diffusion to the cell wall strongly influence the acoustic behavior of the cell. Using the relaxation time of the ν4 vibrational mode of CF4 as a fitting parameter its value was determined to be three times slower than VT relaxation from the ν2 level. The experimental values for the response were predicted by the model with an error of less than 10% in the whole pressure range. Predictions for the optoacoustic signal for different resonator dimensions were also confirmed. Model predictions for the optoacoustic signal for mixtures of gases and the influence of the temperature are also given. Especially the option of cooling the gas seems to be attractive for the case of CF4

CF4 combination band absorption spectroscopy

Absorption and linewidth measurements of the R+ (29) (ν2 + ν4) A14 + E9 + F114 transition in CF4 are reported as a function of gas pressure and temperature. From these a value for the Einstein A coefficient (0.0185 ± 0.0010 sec−1) is deduced. A model for low temperature absorption and a detailed analysis of additional absorption lines are given. The Doppler width reported by Eckhardt et al. (J. Mol. Spectrosc.90, 321–326 (1981)) at 150 K is confirmed, while the homogeneous linewidth is found to be 10% smaller. The frequency displacement of the absorption feature from the CO2 9R(12) emission line center is found to be (34 ± 2) MHz independent of temperature and pressure as may be expected. The room temperature absorption measurements of Radziemski et al. (Opt. Lett.3, 241–243 (1978)) at three different pressures are confirmed but the absorption is not due to a single line and therefore the corresponding dipole moment calculated from those data (Proc. Soc. Photo-Opt. Instrum. Eng.288, 209–216 (1981)) is too large

A parametric study of the output of the optically pumped continuous wave CF4 laser

A parametric study of laser output versus CF4 pressure and temperature was performed and correlated with a model for the gain in the system, which includes the relevant relaxation processes. Lasing in CF4 was observed at temperatures below 170 K. Cooling the CF4 gas, the output power of the laser increased from 3 mW at 142 K to 5 mW at 113 K, when 4% of the radiation was coupled out. Chopping the pump, the 16-μm signal consisted of a peak decaying in approximately 2 ms, superimposed on a CW background. This decay is caused by the slow relaxation in the CF4 laser, resulting in filling of the lower laser level. For the CW CF4 laser, vibrational relaxation from the laser lower level is even slower than diffusion to the cold cell walls. To increase the relaxation rate, HD was added. In this molecule, the J=1→3 rotational transition at 447 cm-1 is almost resonant with the ν2 vibration in CF4. Maximum CW output was increased by 25% in a mixture containing 10% HD. At the same time, the lasing pressure range was extende

A Yohimbine-Dependent, UK14,304 Induced Ion Transient in HT29 Cells Studied by X-Ray Microanalysis

The response of HT29 cells to α2-adrenoceptor agonists was investigated by X-ray microanalysis. Treatment of the cells with 10 μM UK14,304, an α2-agonist, resulted in a rise of intracellular Cl and Na, suggesting an antisecretory effect. When the cells were exposed to yohimbine or RX821002, both of which are arantagonists, the former had no effect, whereas the latter produced a minor decrease of the cellular Na, Cl and K concentrations. However, when the cells were pretreated with yohimbine, UK14,304 induced a transient change in ion concentrations, which consisted of a rapid increase of intracellular Cl, Na and a decrease of K at 1 and 5 minutes. Concurrently, a marked increase in intracellular Ca was found. This transient change could also be induced by different doses of UK14,304 (100 nM or 10 μM) after preincubation with yohimbine (0.3 μM or 3 μM). In contrast, UK14,304 did not elicit a similar response after pretreatment of the HT29 cells with RX821002. Also, yohimbine did not affect the ion content of the cells after pretreatment with UK14,304. These results indicate that yohimbine may have a specific effect by which the cells are sensitized to the ion transient induced by UK14,304

Post-Mortem Storage of Tissue for X-Ray Microanalysis in Pathology

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°C or even at -20°C retained the elemental distribution at the cellular level, and also retained the difference between a \u27normal\u27 and a \u27pathological\u27 (mimicked by an inject ion of a high dose of isoproterenol) 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