Properties of frozen sections relevant to quantitative microanalysis

Difficulties in the quantitative X-ray microanalysis of frozen sections may conceivably arise from ice-crystal damage and from electron-beam damage. X-ray peak-to-continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice-crystal formation existing prior to sublimation. The experiments in these reports are re-interpreted here; it is argued that peak intensities may be affected by ice-crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum. The accuracy of the peak-to-continuum method is affected by beam-induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold-stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen-hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen-hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss. Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X-ray analysis at very high resolution.     

An interactive image analysis system for mean particle volume estimation using stereological principles

A semi-automatic image analysis system based on the stereological approach of point-sampled intercepts for mean volume estimation is described. The method used requires no assumptions about particle geometries and is thus general in its application. To ease implementation in this application, the assumption of particle convexity is made, making the measurement of intercepts easier for the user. The system was tested with computer-generated sample sections to determine the accuracy and precision of the estimates obtained. The results were found to be within 10% of expected values in all cases. When tested with ‘real’ samples a typical result gave a coefficient of variation of 9% from eight independent estimates of one sample.     

Three-dimensional reconstruction from serial section images by computer graphics

A method is described that allows a three-dimensional object to be reconstructed from micrographs of serial sections by means of computer graphics. The reconstructed object, which can be rotated three-dimensionally, is displayed on a colour visual display unit, and the object is shaded in order to provide an illusion of a three-dimensional structure. Moreover, the technique makes it possible to observe an inner structure as seen through an outer one.     

Two-phase flow in porous media: obtaining sharp digitized images of serial sections for subsequent quantitative analysis

Microscopic studies of the distributions of oil and aqueous phases within the pore space of reservoir rocks, at various relative saturations, are of fundamental importance in understanding migration, distribution, mobilization and recovery of oil in the context of a particular (enhanced) oil recovery method. The most effective approach to use for these studies is three-dimensional computer reconstruction with the data for the three-phase system (rock, oil, aqueous phase) entered by video digitization of a set of serial sections. A novel technique for creating accurate three-phase digitized images from the serial section data is described. By use of appropriate pore cast materials and lighting conditions, three different two-phase video images are created and digitized. In each image, a different one of the three phases in the pore cast is one of the two distinguishable phases. The two best two-phase digitized images are combined to give the three-phase digitized image.     

A new heavy-metal-containing resin for low-temperature embedding and imaging of unstained sections of biological material

A styrene-based, low viscosity, negative-staining resin has been formulated for low-temperature embedding of unstained biological material. The resin, containing 3 atom% tin, permits sample preparations at 243°K, and provides sufficient contrast with bright field conventional transmission electron microscopy (CTEM) and excellent contrast in scanning transmission electron microscopy (STEM) ratio mode.     

Quantitative X-ray microanalysis of biological specimens

Qualitative X-ray microanalysis of biological specimens requires an approach that is somewhat different from that used in the materials sciences. The first step is deconvolution and background subtraction on the obtained spectrum. The further treatment depends on the type of specimen: thin, thick, or semithick. For thin sections, the continuum method of quantitation is most often used, but it should be combined with an accurate correction for extraneous background. However, alternative methods to determine local mass should also be considered. In the analysis of biological bulk specimens, the ZAF-correction method appears to be less useful, primarily because of the uneven surface of biological specimens. The peak-to-local background model may be a more adequate method for thick specimens that are not mounted on a thick substrate. Quantitative X-ray microanalysis of biological specimens generally requires the use of standards that preferably should resemble the specimen in chemical and physical properties. Special problems in biological microanalysis include low count rates, specimen instability and mass loss, extraneous contributions to the spectrum, and preparative artifacts affecting quantitation. A relatively recent development in X-ray microanalysis of biological specimens is the quantitative determination of local water content.     

Surface areas of basolateral membranes in renal distal tubules estimated by vertical sections

The surface area of the Na, K-ATPase-rich basolateral membranes in the medullary thick ascending limb of the distal tubule in the rabbit kidney was determined stereologically using the method of ‘vertical sections’, whereby unbiased surface estimates are obtained by imposing a cycloid test-lattice on micrographs of ultrathin sections cut roughly longitudinal to the tubular axis. The unbiased estimate of the surface area of basolateral membranes per tubule length in the thick ascending limb was 1.45 × 10⁶ μm²/mm. The results are compared with previous surface area measurements in this segment of the tubule and discussed with respect to the contributions from all sampling levels to the real biological variation. An optimized sampling scheme with a roughly fourfold reduction in workload is suggested.     

Using linear sections instead of uniform ones to analyze the coupled nonuniform transmission lines

In this article, a new method is introduced to frequency domain analyze arbitrary coupled nonuniform transmission lines (CNTLs). The CNTLs are subdivided into several short linear sections instead of uniform sections. The chain parameter matrix of linear sections is obtained by expressing the voltages and currents by power series expansion. This method is applicable to all arbitrary CNTLs. The accuracy of the proposed method is verified using a comprehensive example. © 2008 Wiley Periodicals, Inc. Int J RF and Microwave CAE, 2009.