Analysis of nerve fibers and their distribution in histologic sections of the human brain

The field of quantitative analysis and subsequent mapping of the cerebral cortex has developed rapidly. New powerful tools have been applied to investigate large regions of complex folded gyrencephalic cortices in order to detect structural transition regions that might partition different cortical fields of disjunct neuronal functions. We have developed a new mapping approach based on axoarchitectonics, a method of cortical visualization that previously has been used only indirectly with regard to myeloarchitectonics. Myeloarchitectonic visualization has the disadvantage of producing strong agglomerative effects of closely neighbored nerve fibers. Therefore, single and neurofunctional-relevant parameters such as axonal branchings, axon areas, and axon numbers have not been determinable with satisfying precision. As a result, different staining techniques had to be explored in order to achieve a suitable histologic staining for axon visualization. The best results were obtained after modifying the Naoumenko-Feigin staining for axons. From these contrast-rich stained histologic sections, videomicroscopic digital image tiles were generated and analyzed using a new fiber analysis framework. Finally, the analysis of histologic images provided topologic ordered parameters of axons that were transferred into parameter maps. The axon parameter maps were analyzed further via a recently developed traverse generating algorithm that calculated test lines oriented perpendicular to the cortical surface and white matter border. The gray value coded parameters of the parameter maps were then transferred into profile arrays. These profile arrays were statistically analyzed by a reliable excess mass approach we recently developed. We found that specific axonal parameters are preferentially distributed throughout granular and agranular types of cortex. Furthermore, our new procedure detected transition regions originally defined by changes of cytoarchitectonic layering. Statistically significant inhomogeneities of the distribution of certain axon quantities were shown to indicate a subparcellation of areas 4 and 6. The quantification techniques established here for the analysis of spatial axon distributions within larger regions of the cerebral cortex are suitable to detect inhomogeneities of laminar axon patterns. Hence, these techniques can be recommended for systematic and observer-supported cortical area mapping and parcellation studies.     

Digital image tiles: a method for the processing of large sections

Segmentation of large areas of light microscopic slides into N by N fields, and each of these fields into M digital image tiles, allows the scanning, storage and digital processing of large images. Any of the original N² fields or composites of M adjacent tiles can be recalled to the video display for analysis. Developed procedures for use on a microscope equipped with a precision scanning stage allow registration of the image coordinates (X-Y) for any original or composite field and the alignment of one of these fields along the depth (Z) axis by means of external, machined fiducial marks in serial sections. To facilitate work whenever unavoidable, we have incorporated methods for digital image panning and zooming (changes of magnification) and discuss their use and implications.     

Three-dimensional reconstruction of a plant dictyosome from series of ultrathin sections using computer image processing

A single dictyosome from an actively secreting ovary gland cell of Aptenia cordifolia has been reconstructed in 3-D from a series of twenty-nine electron micrographs by computer image processing. The reconstruction is presented under different viewing angles in the form of shaded perspective displays. From these displays the entire dictyosome, surrounded by numerous vesicles, appears to be more a spherical than a flat body. The plate-like region of the dictyósome is demonstrated when only a portion of the electron micrographs is used for the image processing, leading to ‘cut-off’ displays. Since some upper planes were removed, such ‘cut-off’ displays revealed both tubular connections between cisternae of the dictyosome and the neighbouring endoplasmic reticulum as well as tubular continuities between adjacent Golgi cisternae within the same stack. Possible consequences of both types of interconnections on transport and processing of proteins and glycoproteins are discussed.     

Determination of areal densities of blood vessel wall components in histological sections by means of image analysis

A technique is described which allows quantification of specifically stained, intermingled vessel wall components in paraffin-embedded tissue. The dissociation of elastic, muscular and connective tissue and the determination of their areal densities in the superior vena cava and in the ascending aorta of the dog, was performed by automated image analysis. The percentages of elastic+connective tissue, determined by this technique, correlated significantly with the data derived from biochemical measurements. It was further found that the structure of the vena cava wall was a function of the distance from the right atrium.     

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

Light microscopic autoradiographs of H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.     

Computerized morphometry of the cirrhotic liver: comparative analysis in primary biliary cirrhosis, alcoholic cirrhosis, and posthepatitic cirrhosis

Fibrosis and nodular regeneration are the hallmarks of liver cirrhosis. To assess the degree of fibrosis and the severity of the structural changes affecting parenchymal and extraparenchymal components in liver cirrhosis, a computerized morphometric model has been applied to liver specimens from patients undergoing liver transplantation for primary biliary cirrhosis, posthepatitic and alcoholic cirrhosis. Fifty-eight hepatectomy specimens from patients undergoing liver transplantation for cirrhosis were analyzed: 17 alcoholic, 28 posthepatitic (HBV-related and HCV-related cirrhosis), and 13 primary biliary cirrhoses. Liver specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with chromotrope-aniline blue method and monoclonal antibodies against cytokeratin 7 and CD31. Volume fractions of parenchymal compartment and fibrosis were stereologically determined on the specimens stained with chromotrope-aniline blue method. Volume fractions of portal bile ducts, proliferated bile ductules, and hepatocytes with biliary metaplasia were measured on cytokeratin 7 stains, while volume fractions of capillary units have been evaluated on CD31 staining. Volume fraction of fibrosis was higher in primary biliary cirrhosis than in the other disease-induced cirrhosis. The main differences were related to immunohistochemical staining. Volume fraction of hepatocytes with biliary metaplasia was higher in HCV-related cirrhosis, whereas volume fractions of biliary structures were more prominent in HBV-related cirrhosis. Primary biliary cirrhosis was characterized by a reduced number of bile ducts and by a wider expression of cytokeratin 7 into periportal hepatocytes. Capillary units were more prominent in primary biliary cirrhosis than alcoholic and posthepatitic cirrhosis. Our computerized morphometric model well describes and quantifies the morphological alterations of the liver and it could represent an adjunctive tool to evaluate the degree of dysplastic phenomena involving parenchymal and extraparenchymal compartments.     

Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3-D reconstruction

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true-scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.     

Three-dimensional morphometry in scanning electron microscopy: a technique for accurate dimensional and angular measurements of microstructures using stereopaired digitized images and digital image analysis

A method for accurate dimensional and angular measurements of microstructures analysed in the scanning electron microscope is described. The method considers central and parallel projections and involves (a) digital image acquisition of stereopaired images from the scanning electron microscope's photodisplay, (b) generation of 3D-image representations, (c) setting of measuring points in the digitized stereopaired images, (d) computation of exact space coordinates ( x / y / z ) from the corresponding point coordinates ( x _L/ y _L; x _R/ y _R), (e) determination of distances and angles between consecutive corresponding points using vector equations, and (f) transfer of computed data into spreadsheets of the data analysis software using dynamic data exchange with simultaneous graphical display of the frequency distribution of variables.
Measurements performed on specimens with known dimensions (grid with 10 μm wide square meshes, polystyrene beads with 0.33 μm diameter) and angles (synthetic crystals of K(Al,Cr)[SO₄], CuSO₄.5H₂O and NaCl) revealed a high accuracy in dimensional as well as angular measurements (total error 1 ± 0.5%).
In Monte Carlo experiments the overall error was found to depend strongly on the size of the measured structure relative to the size of the measurement field (field width).     

Reproducibility and accuracy of interactive segmentation procedures for image analysis in cytology

The segmentation of nuclear images is a crucial step in the development of procedures using image analysis for the cytological diagnosis of cancer. The purpose of this study is to evaluate the reproducibility and accuracy of several interactive segmentation methods which can be used in this context. Four methods were studied: a thresholding-based method enabling selection of intensity histogram contrast and brightness, manual tracing with a stylus, and arc- and ellipse-fitting routines. Features of nuclear size and shape were derived from nuclei segmented on repeated occasions by several individuals. Variance component models provided a statistical framework for evaluating the intraobserver and interobserver variability of these measurements in terms of their intraclass correlation coefficients. Of the methods tested, the arc-fitting segmentation method gave the most reproducible results, and thresholding the least. Reproducibility was generally very high both between individuals and for repeated segmentations by a single individual. Accuracies of area measurements for the various methods, as determined with respect to point counting, paralleled the reproducibilities of the methods. Sample size requirements were observed to be more dependent on the biological variability of the tissue sampled than on the particular segmentation method or on the number of individuals performing segmentation.     

Confocal microscopy and image processing in the study of plant nuclear structure

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide-field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three-dimensional (3-D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3-D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.     

Precision of Cavalieri sections and slices with local errors

Cavalieri sections — and more recently Cavalieri slices, especially in combination with non-invasive scanning — are widely used to estimate volumes. Physical Cavalieri slices are also increasingly used to estimate neuron numbers via the optical fractionator. In either case, the prediction of the error variance is important to assess optimal sample sizes. The error variance consists of two components, one due to the variation among the true contents of sections or slices, and the other due to local or ‘nugget’ errors. The latter may arise for instance when estimating section areas by point counting, or when counting discrete particles in slices or disectors. In this paper, a fairly comprehensive set of prediction formulae is presented to separate both variance components.     

Numerical simulation of auto-regulation and collateral circulation in the human brain

A novel approach using computational fluid dynamics (CFD) and magnetic resonance image (MRI) is applied to model the auto-regulation and blood flow in the human brain. To provide a basic understanding of the auto-regulation mechanism in the brain, an anatomical Circle of Willis configuration is reconstructed from subject-specific magnetic resonance images using image segmentation methods and grid generation techniques. The three-dimensional unsteady incompressible Navier-Stokes equations are solved iteratively using the pseudocompressibility method and dual time stepping method. For the efficient simulation of three-dimensional time-dependent flows, parallel compulations based on a domain decomposition method are performed. A simple auto-regulation algorithm is presented to model the dynamic peripheral resistance due to arteriolar contraction and dilatation. The present numerical methods are then used to simulate the auto-regulation of blood flow in the realistic Circle of Willis model with geometrical variants. The computed results show the correlation between abnormal vascular structures and the auto-regulation mechanism in the cerebral circulation.     

Image analysis of Nissl-stained neuronal perikarya in the primary visual cortex of the rat: Automatic detection and segmentation of neuronal profiles with nuclei and nucleoli*

An image analysing procedure for the morphometric characterization of cortical neurons in Nissl-stained brain sections is described. It consists of the automatic detection of cellular profiles and their compartments: cytoplasm, nucleus and nucleolus. The algorithm was designed to cope with the large morphological spectrum of cortical perikarya (e.g. geometrical properties of perikarya, staining intensities of cell compartments and nucleo-plasmic area-ratio) including pyramidal (Golgi-category I) and non-pyramidal (Golgi-category II) neurons. Clusters of cells were separated and non-neuronal structures (e.g. glia, endothelial cells) as well as tangential, non-nucleolated sections through neuronal perikarya recognized and excluded from further analysis without requiring interactive procedures. The performance of the profile recognition procedure was evaluated using 426 nucleolated and non-nucleolated profiles of different types of neurons in the primary visual cortex of the rat. Nucleolated profiles were recognized as such with a 91% accuracy, non-nucleolated profiles were rejected correctly in 90% of cases. After automatic segmentation and selection of nucleolated neuronal profiles from the microscopic field, a large set of quantitative morphological features including geometrical, densitometrical and textural parameters can be measured using high power light microscopy. This permits quantitative morphometric characterization of different neuronal types. This procedure is the first part of a system for the automatic classification of Nissl-stained cortical neurons.     

Second-order stereology of benign and malignant alterations of the human mammary gland

The purpose of the present study was a quantitative characterization of the three-dimensional arrangement of the epithelial component of benign and malignant alterations of the female breast by combining stereology with stochastic geometry. Twenty cases of fibrous mastopathy and 20 cases of invasive ductal mammary cancer were studied at the light microscopic level. Segmentation of the epithelial tissue component was performed with an image analyser. From the resulting binary images, unbiased estimates of the covariance C(r) and the intensity V_v of the epithelial volume component were obtained automatically by computer. From these data, estimates of the correlation function k(r), of the pair correlation function g(r), of the radial distribution function RDF(r) and of the reduced second moment function K(r) of epithelial volume were determined. The estimates of C(r) and RDF(r) differed between groups, but these functions depend on spatial pattern and V_v. As carcinomas showed a significantly higher epithelial volume density V_v than mastopathies, estimation of C(r) and RDF(r) alone did not permit a safe distinction between possibly different types of spatial arrangement of epithelium in the benign and malignant lesions. Analysis of the estimates of k(r), g(r) and K(r), which are not influenced by V_v, showed definite interaction between epithelial volume elements, with clustering at short distances and repulsion at long distances. In both groups, the null hypothesis of purely random arrangement of epithelium had to be rejected. The clearest distinction between groups was obtained by estimation of g(r), which showed that short-range, tubular pattern as well as long-range, lobular architecture are better preserved in benign than in malignant lesions. The low interindividual scatter of k(r), g(r) and K(r) indicates a high biological significance of spatial pattern, which is presumably under strict genetic control. Potential uses of the method are: (i) the identification of biomathematical models which could contribute to a better understanding of the growth processes involved, (ii) conditional simulation of the underlying three-dimensional structures by computer, and (iii) supporting the diagnosis of mammary lesions with borderline histopathological appearance.     

Extending the dynamic range of fibre length and fibre aspect ratios by automated image analysis

An automatic image analysis system has been developed, that is capable of scanning a large sample area (70 mm × 70 mm) using transmitted light microscopy. The analysis, based on transparent algorithms, successfully discriminates between overlapping and touching fibres, so that sample preparation time is minimized. The system utilizes overlapping image frames to reconstruct partial fibre images and impart unique fibre length information in the range micrometres to millimetres. The software has been made adaptable to enable the accurate measurement of fibre lengths for fibres that curve over many millimetres. Data are presented illustrating the accurate measurement of fibre lengths over a large range of fibre aspect ratios.     

Application of recording and processing of energy-filtered image sequences for the elemental mapping of biological specimens: Imaging-Spectrum

Computerized energy-filtered transmission electron microscope (EFTEM) permits the recording and the processing of energy-filtered images, allowing a part of an electron energy-loss spectrum for each picture element to be obtained. This method, called ‘Imaging-Spectrum’, uses a Zeiss CEM902 coupled to several image analysis systems. The actual configuration records sequences of 48 images, 256 × 256 pixels, in steps of the energy loss, ΔE. Processing these sequences results in part of a core-loss EELS-spectrum for each pixel. This approach produces elemental maps with a short processing time. We have implemented three kinds of background calculation for the image subtraction. The influence of the irradiation dose and of the energy selecting slit width on the quality of the spectra is investigated. The method is applied to the analysis of some biological specimens (pericellular coat behaviour during adhesion between macrophages and red blood cells and location of calcite microcrystals in dental pulp cells). The Imaging-Spectrum method appears to be suitable for the analysis of large areas.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20–50 and 1–2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA–DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1–2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.