Assessment of the possibilities of the dual-energy X-ray absorptiometry of multicomponent samples with variable content

Possibility of the employment of a linear coupling equation of mass attenuation coefficients for two energies for the density determination by dual-energy X-ray absorptiometry of multicomponent samples with variable content was theoretically approved and experimentally demonstrated. The relative error of the density measurements of aqueous-salt and aqueous-alcoholic solutions is 0.5% with a maximum change of the mass attenuation coefficient for the K _α line of zirconium being 70%.     

EVALUATION OF SELF-ATTENUATION COEFFICIENTS OF OIL-PRODUCED SCALES FOR GAMMA-RAY SPECTROSCOPIC ANALYSIS

This study focuses on the evaluation of self-attenuation correction factors (C_f) and linear?attenuation coefficients (μ) of scale samples, produced from the oil fields. This was performed using ¹⁵²Eu point source at the optimal geometry found (i.e., 3 mm). The scale samples were of apparent density ranging 1.04 ? 3.08 g cm^?3. The results illustrated that self-attenuation correction values were as high as 5.08 for the most dense samples at γ-energy line of 45.5 keV. High correlation was observed by Pearson matrix between self-attenuation correction factor and sample density (correlation coefficient of 0.967 at γ-energy of 45.5 keV). A simplified model of the relationship between these variables was proposed. The remarkable finding was that when scale sample density increased self-absorption extended to occur to a higher energy line. Hence, self-attenuation correction was negligible at γ-energies of 122, 222, and 344 keV for samples with density ranging 1.04–1.41, 1.45–2.04, and 2.12–3.08 g cm^?3, respectively. Since linear attenuation coefficient is material sensitive, it was calculated for each sample. Analysis of variance (ANOVA) reflected the linear relationship between linear-attenuation coefficient and sample density up to 344 keV which disappeared thereafter. The data obtained allowed an accurate determination of the concentrations of γ-emitters in scale samples within the energy range of 45.5–1408 keV.     

Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive x-ray spectrometry

Backscattered electron (BSE) imaging has proven to be a useful method for analyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has suggested that BSE images can be standardized by energy-dispersive x-ray spectrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methyl-methacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.     

Single x-ray transmission system for bone mineral density determination

Bones are the support of the body. They are composed of many inorganic compounds and other organic materials that all together can be used to determine the mineral density of the bones. The bone mineral density is a measure index that is widely used as an indicator of the health of the bone. A typical manner to evaluate the quality of the bone is a densitometry study; a dual x-ray absorptiometry system based study that has been widely used to assess the mineral density of some animals' bones. However, despite the success stories of utilizing these systems in many different applications, it is a very expensive method that requires frequent calibration processes to work properly. Moreover, its usage in small species applications (e.g., rodents) has not been quite demonstrated yet. Following this argument, it is suggested that there is a need for an instrument that would perform such a task in a more reliable and economical manner. Therefore, in this paper we explore the possibility to develop a new, affordable, and reliable single x-ray absorptiometry system. The method consists of utilizing a single x-ray source, an x-ray image sensor, and a computer platform that all together, as a whole, will allow us to calculate the mineral density of the bone. Utilizing an x-ray transmission theory modified through a version of the Lambert-Beer law equation, a law that expresses the relationship among the energy absorbed, the thickness, and the absorption coefficient of the sample at the x-rays wavelength to calculate the mineral density of the bone can be advantageous. Having determined the parameter equation that defines the ratio of the pixels in radiographies and the bone mineral density [measured in mass per unit of area (g/cm(2))], we demonstrated the utility of our novel methodology by calculating the mineral density of Wistar rats' femur bones.     

Mineral content changes in bone associated with damage induced by the electron beam

Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200x embedded, 200x unembedded, and 1000x embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200x, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200x, 1000x data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.     

Mean atomic number and backscattered electron coefficient calculations for some materials with low mean atomic number

In modelling electron backscattering from solids using Monte Carlo simulations, knowledge of mean atomic number, mean atomic weight, and density of the bulk material are required. We studied four different ways in common useforthe calculation of mean atomic number. An alternative and improved approach is to calculate the mean backscattered electron (BSE) coefficient, η , from a knowledge of the elemental composition and values of η for the elements. Again, we studied a number of formulae suggested for this averaging process. As it is not possible to measure η directly for a number of elements, the method used to interpolate between elements with known η was also examined. In addition, we obtained experimental backscattering relationships for topography-free samples of poly (methylmethacrylate) (PMMA), carbon, aluminium, and a series of novel halogenated resins, all solids with relatively low mean atomic numbers, and calcified tissues. None of the methods for determining mean atomic number placed the materials of interest in the correct sequence of their experimentally determined BSE peaks: the data differed widely between the individual methods. The averaged BSE coefficient calculated by the Castaing formula gave the best agreement with the experimentally derived data.     

Limitations of using micro-computed tomography to predict bone-implant contact and mechanical fixation

Fixation of metallic implants to bone through osseointegration is important in orthopaedics and dentistry. Model systems for studying this phenomenon would benefit from a non-destructive imaging modality so that mechanical and morphological endpoints can more readily be examined in the same specimens. The purpose of this study was to assess the utility of an automated microcomputed tomography (μCT) program for predicting bone-implant contact (BIC) and mechanical fixation strength in a rat model. Femurs in which 1.5-mm-diameter titanium implants had been in place for 4 weeks were either embedded in polymethylmethacrylate (PMMA) for preparation of 1-mm-thick cross-sectional slabs (16 femurs: 32 slabs) or were used for mechanical implant pull-out testing (n= 18 femurs). All samples were scanned by μCT at 70 kVp with 16 μm voxels and assessed by the manufacturer's software for assessing 'osseointegration volume per total volume' (OV/TV). OV/TV measures bone volume per total volume (BV/TV) in a 3-voxel-thick ring that by default excludes the 3 voxels immediately adjacent to the implant to avoid metal-induced artefacts. The plastic-embedded samples were also analysed by backscatter scanning electron microscopy (bSEM) to provide a direct comparison of OV/TV with a well-accepted technique for BIC. In μCT images in which the implant was directly embedded within PMMA, there was a zone of elevated attenuation (>50% of the attenuation value used to segment bone from marrow) which extended 48 μm away from the implant surface. Comparison of the bSEM and μCT images showed high correlations for BV/TV measurements in areas not affected by metal-induced artefacts. In addition for bSEM images, we found that there were high correlations between peri-implant BV/TV within 12 μm of the implant surface and BIC (correlation coefficients ≥0.8, p < 0.05). OV/TV as measured on μCT images was not significantly correlated with BIC as measured on the corresponding bSEM images. However, OV/TV was significantly, but weakly, correlated with implant pull-out strength (r= 0.401, p= 0.049) and energy to failure (r= 0.435, p= 0.035). Thus, the need for the 48-μm-thick exclusion zone in the OV/TV program to avoid metal-induced artefacts with the scanner used in this study means that it is not possible to make bone measurements sufficiently close to the implant surface to obtain an accurate assessment of BIC. Current generation laboratory-based μCT scanners typically have voxel sizes of 6-8 μm or larger which will still not overcome this limitation. Thus, peri-implant bone measurements at these resolutions should only be used as a guide to predict implant fixation and should not be over-interpreted as a measurement of BIC. Newer generation laboratory-based μCT scanners have several improvements including better spatial resolution and X-ray sources and appear to have less severe metal-induced artefacts, but will need appropriate validation as they become available to researchers. Regardless of the μCT scanner being used, we recommend that detailed validation studies be performed for any study using metal implants because variation in the composition and geometry of the particular implants used may lead to different artefact patterns.     

Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.     

Sample preparation and microscopical investigation techniques for metal and ceramics containing graphite and graphene‐like layered particles

Scanning electron microscopy (SEM) techniques are widely used in microstructural investigations of materials since it can provide surface morphology, topography, and chemical information. However, it is important to use correct imaging and sample preparation techniques to reveal the microstructures of materials composed of components with different polishing characteristics such as grey cast iron, graphene platelets (GPLs)‐added SiAlON composite, SiC and B₄C ceramics containing graphite or graphene‐like layered particles. In this study, all microstructural details of gray cast iron were successfully revealed by using argon ion beam milling as an alternative to the standard sample preparation method for cast irons, that is, mechanical polishing followed by chemical etching. The in‐lens secondary electron (I‐L‐SE) image was clearly displayed on the surface details of the graphites that could not be revealed by backscattered electron (BSE) and Everhart–Thornley secondary electron (E‐T SE) images. Mechanical polishing leads to pull‐out of GPLs from SiAlON surface, whereas argon ion beam milling preserved the GPLs and resulted in smooth surface. Grain and grain boundaries of polycrystalline SiC and B₄C were easily revealed by using I‐L SE image in the SEM after only mechanical polishing without any etching process. While the BSE and E‐T SE images did not clearly show the residual graphites in the microstructure, their distribution in the B₄C matrix was fully revealed in the I‐L SE image.     

The effect of Al2O3 on the friction performance of automotive brake friction materials

This study consists of two stages. In the first stage, bronze-based break linings were produced and friction-wear properties of them were investigated. In the second stage, 0.5%, 1%, 2% and 4% alumina (Al₂O₃) powders were added to the bronze-based powders and Al₂O₃ reinforced bronze-based break linings were produced. Friction–wear properties of the Al₂O₃ reinforced samples were aslo investigated and compared to those of plain bronze-based ones. For this purpose, friction coefficient and wear behaviour of the samples were tested on the grey cast iron disc. The hardness and density of the samples were also determined. Microstructures of the samples before and after the sintering and the worn surfaces of the wear specimens were examined using a scanning electron microscope (SEM). The sample compacted at 350 MPa and sintered at 820 °C exhibited the optimum friction–wear behaviour. With increase in friction surface temperature, a reduction in the friction coefficient of the samples was observed. The lowest reduction in the friction coefficient with increasing temperature was for the 2% and 4% Al₂O₃ reinforced samples. The SEM images of the sample indicated that increase in Al₂O₃ content resulted in adhesive wear. With increase in Al₂O₃ content, a reduction in mass loss of the samples was also observed. Overall, the samples reinforced with 2% and 4% Al₂O₃ exhibited the best results.     

Influence of topography and specimen preparation on backscattered electron images of bone

Backscattered electron (BSE) images of bone exhibit graylevel contrast between adjacent lamellae. Mathematical models suggest that interlamellar contrast in BSE images is an artifact due to topographic irregularities. However, little experimental evidence has been published to support these models, and it is not clear whether submicron topographical features will alter BSE graylevels. The goal of this study was to determine the effects of topography on BSE image mean graylevels and graylevel histogram widths using conventional specimen preparation techniques. White-light interferometry and quantitative BSE imaging were used to investigate the relationship between the BSE signal and specimen roughness. Backscattered electron image graylevel histogram widths correlated highly with surface roughness in rough preparations of homogeneous materials. The relationship between BSE histogram width and surface roughness was specimen dependent. Specimen topography coincided with the lamellar patterns within the bone tissue. Diamond micromilling reduced average surface roughness when compared with manual polishing techniques but did not significantly affect BSE graylevel histogram width. The study suggests that topography is a confounding factor in quantitative BSE analysis of bone. However, there is little quantitative difference between low-to-moderate magnification BSE images of bone specimens prepared by conventional polishing or diamond micromilling.     

A fast experimental beam hardening correction method for accurate bone mineral measurements in 3D μCT imaging system

Bone mineral density plays an important role in the determination of bone strength and fracture risks. Consequently, it is very important to obtain accurate bone mineral density measurements. The microcomputerized tomography system provides 3D information about the architectural properties of bone. Quantitative analysis accuracy is decreased by the presence of artefacts in the reconstructed images, mainly due to beam hardening artefacts (such as cupping artefacts). In this paper, we introduced a new beam hardening correction method based on a postreconstruction technique performed with the use of off‐line water and bone linearization curves experimentally calculated aiming to take into account the nonhomogeneity in the scanned animal. In order to evaluate the mass correction rate, calibration line has been carried out to convert the reconstructed linear attenuation coefficient into bone masses. The presented correction method was then applied on a multimaterial cylindrical phantom and on mouse skeleton images. Mass correction rate up to 18% between uncorrected and corrected images were obtained as well as a remarkable improvement of a calculated mouse femur mass has been noticed. Results were also compared to those obtained when using the simple water linearization technique which does not take into account the nonhomogeneity in the object.     

Prediction of cement volume for vertebroplasty based on imaging and biomechanical results

Control of bone cement volume (PMMA) may be critical for preventing complications in vertebroplasty, the percutaneous injection of PMMA into vertebra. The purpose of this study was to predict the optimal volume of PMMA injection based on CT images. For this, correlation between PMMA volume and textural features of CT images was examined before and after surgery to evaluate the appropriate PMMA amount. The gray level run length analysis was used to determine the textural features of the trabecular bone. Estimation of PMMA volume was done using 3D visualization with semi-automatic segmentation on postoperative CT images. Then, finite element (FE) models were constructed based on the CT image data of patients and PMMA volume. Appropriate material properties for the trabecular bone were assigned by converting BMD to elastic modulus. Structural reinforcement due to the changes in PMMA volume and BMD was assessed in terms of axial displacement of the superior endplate. A strong correlation was found between the injected PMMA volume and the area of the intertrabecular space and that of trabecular bone calculated from the CT images (r-0.90 and −0.90, respectively). FE results suggested that vertebroplasty could effectively reinforce the osteoporotic vertebra regardless of BMD or PMMA volume. Effectiveness of additional PMMA injection tended to decrease. For patients with BMD well lower than 50mg/ml, injection of up to 30% volume of the vertebral body is recommended. However, less than 30% is recommended otherwise to avoid any complications from excessive PMMA because the strength has already reached the normal level.     

Quantitative analysis of backscattered-electron contrast in scanning electron microscopy

Backscattered-electron scanning electron microscopy (BSE-SEM) imaging is a valuable technique for materials characterisation because it provides information about the homogeneity of the material in the analysed specimen and is therefore an important technique in modern electron microscopy. However, the information contained in BSE-SEM images is up to now rarely quantitatively evaluated. The main challenge of quantitative BSE-SEM imaging is to relate the measured BSE intensity to the backscattering coefficient η and the (average) atomic number Z to derive chemical information from the BSE-SEM image. We propose a quantitative BSE-SEM method, which is based on the comparison of Monte–Carlo (MC) simulated and measured BSE intensities acquired from wedge-shaped electron-transparent specimens with known thickness profile. The new method also includes measures to improve and validate the agreement of the MC simulations with experimental data. Two different challenging samples (ZnS/Zn(O_xS_1–x)/ZnO/Si-multilayer and PTB7/PC₇₁BM-multilayer systems) are quantitatively analysed, which demonstrates the validity of the proposed method and emphasises the importance of realistic MC simulations for quantitative BSE-SEM analysis. Moreover, MC simulations can be used to optimise the imaging parameters (electron energy, detection-angle range) in advance to avoid tedious experimental trial and error optimisation. Under optimised imaging conditions pre-determined by MC simulations, the BSE-SEM technique is capable of distinguishing materials with small composition differences.     

Microtomographic evaluation of the bone-cell interactions with a silorane-based composite

The low‐shrink Silorane‐based composite could bond effectively to bone and showed the potential be used as a bone cement. Bone organ culture maintains the anatomical order, natural cell‐to‐cell and cell‐to‐matrix relationship. The purpose of this study was to evaluate the responses of bone cells to a Silorane‐based composite which was compared with a representative polymethyl methacrylate (PMMA) bone cement. The critical size defects were created through the parietal bones from one litter of mice. The paired bones were divided into two groups: Silorane‐based composite group and PMMA group. The prepared two groups of disks were put into the defects. The cultures were grown in vitro for 38 days and analyzed with microcomputed‐tomography, dissecting‐microscope, phase‐ contrast‐microscope, scanning‐electron‐microscopy, and energy‐ dispersive‐X‐ray. At the 10th day, the Silorane disk was almost fully covered by a sheet of cells but the cells hardly attached to the disk surface. The edge of the PMMA disk was covered by a sheet of cells and the migrated individual cells attached to the whole surface of the disk. At the 38th day, some cells attached to the exposed disk area of the Silorane disk while the formed tissues covered the whole surface of the PMMA disk. The collagen fibers, globular deposits and bone formation were visible in both groups. The Silorane‐based composite showed promise as a potential bone cement when compared with PMMA which is used in clinical orthopedics. However, the cell attachment to PMMA was evidently better than to Silorane‐based composite. Microsc. Res. Tech. 75:1176–1184, 2012. © 2012 Wiley Periodicals, Inc.     

Study on clear stereo image pair acquisition method for small objects with big vertical size in SLM vision system

Microscopic vision system with stereo light microscope (SLM) has been applied to surface profile measurement. If the vertical size of a small object exceeds the range of depth, its images will contain clear and fuzzy image regions. Hence, in order to obtain clear stereo images, we propose a microscopic sequence image fusion method which is suitable for SLM vision system. First, a solution to capture and align image sequence is designed, which outputs an aligning stereo images. Second, we decompose stereo image sequence by wavelet analysis theory, and obtain a series of high and low frequency coefficients with different resolutions. Then fused stereo images are output based on the high and low frequency coefficient fusion rules proposed in this article. The results show that Δw₁(Δw₂) and ΔZ of stereo images in a sequence have linear relationship. Hence, a procedure for image alignment is necessary before image fusion. In contrast with other image fusion methods, our method can output clear fused stereo images with better performance, which is suitable for SLM vision system, and very helpful for avoiding image fuzzy caused by big vertical size of small objects. Microsc. Res. Tech. 79:408–421, 2016. © 2016 Wiley Periodicals, Inc.     

Two-dimensional measurement of the nonlinearity parameter B/A in excised biological samples

The method previously developed for measuring the acoustic nonlinearity parameter B/A in a liquid sample with a volume as small as 0.1 ml [S. Saito, J. Acoust. Soc. Am. 127, 51(2010)] has been automated and applied to two-dimensional measurements of excised biological samples using a LabVIEW program. The focus of the sound beam is laterally shifted on the 3 × 3 mm(2) area of the sample while measuring the B/A successively. By displaying the result of 256 time repeated measurements with an interval of 0.2 mm in two dimensions, a C-mode image was generated for B/A. The images of linear properties such as density, sound speed, and attenuation coefficient are also obtained. The image, whose pattern can be different from those of the density and sound speed, has the capability to reveal the detailed structure of the B/A, which varies from region to region in a single biological sample. The application of the method to small samples is also demonstrated by measuring a thermally coagulated biological sample.     

Grey theory applied to evaluate the tribological performances of the a-C:H(N) coating films prepared by differing the nitrogen content and the film thickness

The purpose of this paper is to study the tribology performances of the aC:H(N) films by using a nanotester under different scratch loads and velocities. From the measurements of the friction coefficient and wear volume, the tribological performances including wear resistance and friction coefficients were evaluated for the hydrogenated amorphous carbon films prepared by differing film thickness and nitrogen volume friction in the gas mixture of (C₂H₂+N₂). Taguchi experimental design and the grey relational analysis were used to investigate the influence of specimen parameters (film’s thickness, nitrogen content in the film), and operating conditions in tribological tests (scratch load and scratch velocity) on the friction coefficients and the wear volume arising in the specimens with different coating films. It is found that the wear volume of thin film is increased by increasing either the nitrogen volume fraction or film thickness. Moreover, the optimal combination of the testing parameters was also determined in the use of the present model.     

Evaluation of bone strength: correlation between measurements of bone mineral density and drilling force

Bone drilling is a major part of modern orthopaedic surgery which involves the internal fixation of fractured bones. The investigation of bone drilling described in this paper demonstrates the contribution of automation technology towards the study of bone strength. The aim of this preliminary investigation is to establish a relationship between bone drilling forces and measurements of bone mineral density (BMD) by dual energy X-ray absorptiometry (DXA). A linear relationship with a high coefficient of correlation has been found between average drilling forces and BMD measurements at both the greater trochanter and the femoral head of porcine femurs when drilling in the anterior-posterior (AP) direction (i.e. the direction of the DXA scan). It has also been found that in the normal drilling direction (i.e. in the cervical axis direction), which is orthogonal to the DXA scanning direction, there are similar trends between the drilling forces and BMD levels in regions where bone density is more consistent (e.g. the femoral head). The findings of this investigation indicate that analysis of bone drilling forces has the potential to provide useful information about the strength of bone.     

A microcomputer-based system for rapid on-line stereological analysis in the SEM

A microcomputer-based image analysis system for the SEM is described. Through two digital-to-analog convertors it controls the position of the electron beam on the specimen inside the microscope. A raster of 128 × 128 pixels is created. At each pixel the signal from the BSE detector is sampled an operator-defined number of times and the median values of the resulting data streams are stored temporarily in the memory of the computer. Stored images may be redisplayed on the visual and record CRTs of the SEM or saved onto a diskette for subsequent recall and analysis. The 256 grey level image is reduced to one having only 64 grey levels and the distribution of the number of counts falling into any of these bins is displayed on the computer monitor, or output to a plotter or printer. The number of counts in each bin represents stereologically the area (and hence volume) fraction of the sample with that grey level. Three levels of threshold may be applied to give four slices in the image. Area (and hence volume) fractions are then calculated, as is the intercept value between slices. This data is output to a printer. An abbreviated program has also been written to permit greater automation of the analysis of samples of bone. Included is a further reduction in the number of grey levels to 16, and the provision of control for an automatic stage. Although presently being applied to the study of samples of mineralised biological tissues this system could be used for any investigation requiring rapid and simple stereological analysis of specimens in the SEM.