Integration of X-Ray Microanalysis and Morphometry of Biological Material

It was investigated how to extract both morphometrical and X-ray elemental information from scanning electron microscopical (SEM) or scanning transmission electron microscopical (STEM)-images and how to integrate these two information streams either on line or off-line after storage. Cytochemical reaction products in cell organelles in ultrathin sections are the biological structures of interest. In such organelles four different situations can be met: morphologically the structures are homomorph or heteromorph; chemically the elements are distributed either homogeneously or heterogeneously. A new program has been proposed and described, which permits determination of both the area and the mean net-intensity value of chemical elements, inhomogeneously distributed over heteromorph organelles. The value of this integration method is demonstrated by three examples of increasing complexity, starting with two elements which are more or less homogeneously distributed over one lysosome, the establishing of a platinum discontinuity in an acidophilic granule and finally the localization of two chemical elements inhomogeneously distributed over a rather heteromorph phagolysosome. In two examples Chelex ion exchange beads, maximally loaded with the element also present in the structure of interest, are co-embedded with the tissue as internal standards. In such cases the absolute elemental concentration in the structures analysed can be established. The presence of such cross-sectioned beads in the ultrathin sections is also used: 1) to demonstrate their function as models to select the proper conditions for the digital-controlled raster analysis of the unknown cell- or tissue structures, 2) to prove the value of this method

Application of Chelex Standard Beads in Integrated Morphometrical and X-Ray Microanalysis

Chelex ion exchange beads loaded with a known amount (18.3% weight percentage (w/w)) of platinum, have been co-embedded with a mouse peritoneal cell population. To establish the influence of the various deconvolution methods applied, upon the platinum concentration in cytoplasmic granules and erythrocytes these cross-sectioned beads are used as a standard. It is concluded that irrespective of the deconvolution method chosen 1) the Pt concentration inside the particles is identical when the particles and the co-embedded Chelex Pt standard, are analysed strictly under the same instrumental conditions 2) the Pt concentration outside the particle is zero, or virtually zero when that element is absent there (erythrocyte surrounded by Epon) 3) the Pt concentration outside the particle in the surrounding cytoplasm was identical (when the element Pt was present there). The information about the elemental concentration obtained by point analysis in the STEM-mode in homogeneous objects was compared with the mean value obtained by the reduced raster method. The ratio between these values were constant. As an example of a heteromorphic, heterogeneous cell organelle population, the application of the method of integrated morphometrical and chemical (X-ray) analysis is demonstrated on lysosomes within a single human liver parenchymal cell, containing iron and cerium. It was shown that the cerium concentration (from the cytochemical reaction to detect acid phosphatase activity in lysosomes) was rather homogeneously distributed over the small population and in the individual lysosomes. The iron distribution was very inhomogeneous, both in its distribution over the lysosomal area, and among the lysosomes in the population

High-Tc bolometers with silicon-nitride spiderwebsuspension for far-infrared detection

High-Tc GdBa2Cu3O7-δ (GBCO) superconducting transition edge bolometers with operating temperatures near 90 K have been made with both closed silicon-nitride membranes and patterned silicon-nitride (SiN) spiderweb-like suspension structures. As a substrate silicon-on-nitride (SON) wafers are used which are made by fusion bonding of a silicon wafer to a silicon wafer with a silicon-nitride top layer. The resulting monocrystalline silicon top layer on the silicon-nitride membranes enables the epitaxial growth of GBCO. By patterning the silicon-nitride the thermal conductance G is reduced from about 20 to 3 μW/K. The noise of both types of bolometers is dominated by the intrinsic noise from phonon fluctuations in the thermal conductance G. The optical efficiency in the far infrared is about 75% due to a goldblack absorption layer. The noise equivalent power NEP for FIR detection is 1.8 pW/√Hz, and the detectivity D* is 5.4×1010 cm √Hz/W. Time constants are 0.1 and 0.6 s, for the closed membrane and the spiderweb like bolometers respectively. The effective time constant can be reduced with about a factor 3 by using voltage bias. Further reduction necessarily results in an increase of the NEP due to the 1/f noise of the superconductor

Extraneous Background-Correction Program for Matrix Bound Multiple Point X-Ray Microanalysis

A program is described that allows online determination of extraneous background in multiple point X-ray microanalytical matrices. The program is based upon the calculations of the extraneous background for the film (when present), the standard and the unknown by (100 sec.) point analysis. The program searches for a peak-free part of the spectrum in which the calculated value for the extraneous background is about equal to the value in this region of the spectrum (=be). Online the contents of this be-region is subtracted from an unmanipulated continuum region in the vicinity of the element present in the unknown and standard (Pt). During the subsequently performed matrix analysis two arrays are acquired (P-b) and (b-be). From these two arrays, the Rx,st and subsequently the Rx,sp are calculated per pixel, which are converted to (be corrected) concentration arrays. In addition Z2/A-differences between standard and the analyzed specimen are corrected off-line. For each pixel the program judges whether the calculated concentration deviates from the value introduced for the standard. Once differences are registered, adequate corrections are made