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

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

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

A Water-Soluble Iridium Photocatalyst for Chemical Modification of Dehydroalanines in Peptides and Proteins

Dehydroalanine (Dha) residues are attractive non-canonical amino acids that occur naturally in ribosomally synthesised and post-translationally modified peptides (RiPPs). Dha residues are attractive targets for selective late-stage modification of these complex biomolecules. In this work, we show the selective photocatalytic modification of dehydroalanine residues in the antimicrobial peptide nisin and in the proteins Small Ubiquitin-like Modifier (SUMO) and superfolder Green Fluorescent Protein (sfGFP). For this purpose, a new water-soluble iridium(III) photoredox catalyst was used. The design and synthesis of this new photocatalyst, [Ir(dF(CF3 )ppy)2 (dNMe3 bpy)]Cl3, is presented. In contrast to commonly used iridium photocatalysts, this complex is highly water-soluble and allows modification of peptides and proteins in water and aqueous solvents under physiologically relevant conditions and with short reaction times and low reagent and catalyst loadings. This work suggests that photoredox catalysis using this newly designed catalyst is a promising strategy to modify dehydroalanine-containing natural products and thus may have great potential for novel bioconjugation strategies

A Review of New Concepts in Renal Stone Research

Clinical and basic research in the field of urolithiasis has developed rapidly in recent years. Progress in extracorporeal shock wave lithotripsy (ESWL) and percutaneous nephrolithotomy (PNL) has brought about a revolution in the surgical treatment of urolithiasis and research at the cellular and molecular level is now expanding. In spite of these advances, however, clinical treatment of urolithiasis remains far from satisfactory. Stone recurrence in many patients cannot be predicted and is beyond control of urologists mainly because the mechanisms of stone formation are still not fully understood. It is necessary to study the process of stone formation more intensely at the cellular and molecular level, and to strengthen the links between basic and clinical research in the field. In this review, the processes involved in the formation of stones are compared with those involved in normal bio-mineralization and a model of urolithiasis is put forward based on modern systems science. Attention is concentrated on: (a) Directions of research based on physico-chemical theories of stone formation; (b) The role of renal tubular defects in urolithiasis; (c) The role of free radical reactions in stone formation; and (d) Macromolecular abnormalities and their correction

Energy-Filtering Transmission Electron Microscopy of Biological Specimens

By energy-filtering transmission electron microscopy (EFTEM) electrons can be separated by their energy losses. An electron-energy filter, added to the microscope column allows the measurement of the energy distribution of transmitted electrons that have lost energy (\u3c 2,000 eV, with an energy resolution of ~ 1 eV). These filtered electrons, recorded either as a spectrum or as an image, are composed of two parts superimposed on top of each other: (a) the unspecific energy-loss population (= the continuum) and (b) the specific element-related energy-loss population (= the edges). At the edges, electron data in spectra and images are mathematically processed, to obtain the desired element-related net-intensity values or images. These data are related to the total transmitted electron intensity, from the zero-and low-loss spectral region giving the relative spectral-or image intensity ratios (SR*x, IR*x), which can be related to the element concentration. The acquisition of the zero-loss and low-loss data is hampered by the restricted dynamic range of the TV camera. By improvements through the introduction of calibrated attenuation filters in the optical path to the TV-camera, more reliable values for SR*x and IR*x can be acquired. By addition of Bio-standards adjacent to the tissue, a known and unknown concentration of the element present in the same ultrathin section and the bias in the concentration estimation, can be obtained. Some practical examples are given for the estimation of the iron concentration in siderosomes, boron in melanosomes and calcium in calcium oxalate monohydrate crystals

Integrated Image and X-Ray Microanalysis of Hepatic Lysosomes in a Patient with Idiopathic Hemosiderosis Before and After Treatment by Phlebotomy

Morphometrical and X-ray elemental information was extracted from Scanning Transmission Electron Microscopy (STEM) images of hepatic lysosomes of a patient with idiopathic hemosiderosis before and after treatment by phlebotomy. The elements of interest were (i) iron, stored in pathological quantities in hepatic lysosomal structures and (ii) cerium, used as a capture ion after a cytochemical reaction to detect acid phosphatase activity in the lysosomal structures. Morphologically the lysosomal structures are heteromorph and the elements iron and cerium are heterogeneously distributed. With reduced raster (= reduced scanning area) analysis at 16 x 16 pixel points (integrating image and X-ray microanalysis), a marked difference in the area of the cross sectioned lysosomal structures before and after treatment could be demonstrated. Simultaneously the difference in the relative orientation of the elements iron and cerium before and after phlebotomy could be visualized. Chelex ion exchange beads, loaded with 11.5% w/w iron, and coembedded with the tissue blocks, were used as an internal standard. A mean iron peak to background ratio was obtained and a factor, converting ratio to absolute iron concentration, was calculated. The same calculation procedure, now per pixel point, was followed for the hepatic lysosomal structures. A marked difference in iron concentration in the individual lysosomal structures was observed before and after treatment by phlebotomy

Absence of a Transcellular Oxalate Transport Mechanism in LLC-PK1 and MDCK Cells Cultured on Porous Supports

Transepithelial oxalate transport across polarized monolayers of LLC-PK1 cells, grown on collagen-coated microporous membranes in Transwell culture chambers, was studied in double-label experiments using [14C]-oxalate together with [3H]-D-mannitol as an extracellular marker. The [14C]-labeled glucose analog α-methyl-glucoside (α-MG) was used as functional marker for active proximal tubular sugar transport. Cellular uptake of oxalate and α-MG at both the apical and basolateral plasma membrane was determined. When added to the upper compartment, α-MG was actively taken up at the apical membrane, directed through the cells to the basolateral membrane and transported to the lower compartment, indicating functional epithelial sugar transport by LLC-PK1 cells. In LLC-PK1 cells, the uptake of α-MG at the apical membrane was approximately 50 times higher than that at the basolateral membrane. In contrast to this active transport of sugar, LLC-PK1 cells did not demonstrate oxalate uptake either at the apical or basolateral plasma membrane. The apical-to-basolateral (A- \u3e B) flux of oxalate in LLC-PK1 cells was identical to the basolateral-to-apical (B- \u3e A) oxalate flux in these cells. Moreover these flux characteristics were similar to those found for D-mannitol, indicating paracellular movement for both compounds. From these data, it is concluded that, under the experimental conditions used, LLC-PK1 cells do not exhibit a specific transcellular transport system for oxalate

Image Analysis and X-Ray Microanalysis in Cytochemistry

When cytochemical reaction products are homogeneously distributed within an organelle, point analyses suffice for the quantitative approach. However, quantitative analysis becomes tedious, when the elements in the reaction product are inhomogeneously distributed. Problems arise when elements from two reaction products have to be related to each other, or to endogenous cytological products (ferritin, haemosiderin, calcium, electron dense markers), either topographically or in concentration. When analyzing inhomogeneous/heteromorphical reaction product-containing organelles special attention has to be paid to measure and relate both volume and concentration. In this paper a relative simple structure (eosinophil granules) is chosen to demonstrate that the acquisition of the requested morphometrical plus chemical information and their integration is possible. The following points will be covered to acquire the morphometrical and chemical information: a). How to estimate the total cell cross-sectioned area. b). How to estimate the total cross-sectioned area of all reaction product-containing particles inside that cell. The ratio of these two areas will provide the requested information about the particle volume fraction. By using the X-ray detector in addition: c). How to acquire the chemical information at the requested resolution, within a reasonable total acquisition time d). How to integrate the morphometrical and chemical data per organelle, by matrix analysis in a reduced scan area. e). How to acquire quantitative chemical information, by the use of cross-sectioned standards. f). How to make this acquisition method independent from changes in the instrumental conditions during the acquisition