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.     

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.     

Factors which influence light microscopic visualization of biological material in sections prepared for electron microscopy

Epoxy-embedded biological material, sectioned for conventional or high-voltage electron microscopy, can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy. The (phase) contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation, by heavy metal (uranyl and lead) staining and by the section thickness. After screening ultrathin and semithin sections for content with the light microscope, one need stain and examine only those grids containing sections of interest. This approach eliminates the need to screen sections with the electron microscope and, in some cases, the need to stain non-useful sections. This time-saving procedure is particularly useful for studies requiring ultrastructural examination of a selected area or structure which is large enough to be visualized with the light microscope but which comprises only a small volume of the embedded material.     

Two distinct events, section compression and loss of particles ("lost caps"), contribute to z-axis distortion and bias in optical disector counting

Deformation of tissue sections in the z-axis can bias optical disector counting. When samples of particle densities are not representative for the entire tissue section, significant bias of estimated numbers can result. To assess the occurrence, prevalence, extent, sequence of events, and causes of z-axis distortion, the distribution of neuronal nucleoli in thick paraffin and vibratome sections was determined in chicken, rodent, and human brain tissues. When positions of neuronal nucleoli were measured in the z-axis, nucleoli were more frequent at the surfaces (bottom and top) of tissue sections than in the core. This nonlinear z-axis distribution was not lab-, equipment-, or investigator-specific, and was independent of age, fixation quality, coverslipping medium, or paraffin melting temperature, but in paraffin sections, was highly correlated with the tilt of the knife (cutting) angle. Manipulation of subsequent tissue processing steps revealed that two events contribute to z-axis distortion. Initially, a higher density of particles results at surfaces after sectioning, apparently due to section compression. Subsequently, particles can be lost to varying degrees from surfaces during floating or staining and dehydration, resulting in "lost caps." These results may explain different degrees of z-axis distortion between different types of sections and different labs, and reinforce the importance of checking z-axis distributions as a "quality control" prior to selection of guard zones in optical disector counting. Indirect approaches to assess section quality, such as resectioning in a perpendicular plane, yield additional artifacts, and should be replaced by a direct quantitative measurement of z-axis distribution of particles.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Pronounced loss of cell nuclei and anisotropic deformation of thick sections

The local deformation and variations in section thickness are studied in 100-μm thick vibratome sections of well-fixed human brain tissue. During processing, including drying on glass slides, the section thickness is reduced to less than half, but close to the edges there is less shrinkage of the section thickness. Close to both surfaces there is a pronounced reduction in the number of neuronal nucleoli. At the scale of the original section, the upper 15 μm and the lower 10 μm are depleted. The loss is most pronounced at the upper surface, which is unprotected during processing. In the central 70% of the section height, where one would ordinarily use an optical disector for sampling, there is no indication of non-uniform shrinkage. The simplest explanation for the observed loss of nucleoli is that all cells opened by the knife may lose their nuclei across an unprotected section surface. The observations do not generalize to other tissues and other preparation techniques, but illustrate the magnitude of some of the problems for uniform sampling and unbiased estimation in very thick sections. The uniform optical disector sampling of nucleoli in thick sections, as opposed to that of cell nuclei, raises a special problem, which is discussed briefly.     

Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections

Ultrathin sections of rapidly frozen, briefly pre-treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in-vivo order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X-ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M-band and the bridge region of the A-band in great detail. To enhance the detail in the original images, one-dimensional (1-D) and 2-D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one-third of its original value, demonstrated here for the first time is the fact that the characteristic A-band lattice planes are preserved in these sections in 3-D. This confirms the usefulness of cryosections not just for 1-D and 2-D image processing, but also for 3-D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level.     

Immunohistochemistry of neuronal nitric oxide synthase and protein nitration in the striatum of the aged rat

To ascertain the possible implications of the nitric oxide (NO*) producing system in striatal senescence, and by using immunohistochemistry and image-processing approaches, we describe the presence of the enzyme nitric oxide synthase (NOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and nitrotyrosine-derived complexes (N-Tyr) in the striatum of adult and aged rats. The results showed neuronal NOS immunoreactive (nNOS-IR) aspiny medium-sized neurons and nervous fibres in both age groups, with no variation in the percentage of immunoreactive area but a significant decrease in the intensity and in the number of somata with age, which were not related to the observed increase with age of the striatal bundles of the white matter. In addition, NADPH-d activity was detected in neurons with morphology similar to that of the nNOS-IR cells; a decrease in the percentage of area per field and in the number of cells, but an increase in the intensity of staining for the NADPH-d histochemical marker, were detected with age. The number of neuronal NADPH-d somata was higher than for the nNOS-IR ones in both age groups. Moreover, N-Tyr-IR complexes were observed in cells (neurons and glia) and fibres, with a significant increase in the percentage of the area of immunoreaction, related to the increase of white matter, but a decrease in intensity for the aged group. On the other hand, we did not detect the inducible isoform (iNOS) either in adult or in aged rats. Taken together, these results support the contention that NADPH-d staining is not such an unambiguous marker for nNOS, and that increased protein nitration may participate in striatal aging.     

Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing-microscope

A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl-stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing-microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.     

Sampling theory and automated simulations for vertical sections,applied to human brain

In recent years, there have been substantial developments in both magnetic resonance imaging techniques and automatic image analysis software. The purpose of this paper is to develop stereological image sampling theory (i.e. unbiased sampling rules) that can be used by image analysts for estimating geometric quantities such as surface area and volume, and to illustrate its implementation. The methods will ideally be applied automatically on segmented, properly sampled 2D images – although convenient manual application is always an option – and they are of wide applicability in many disciplines. In particular, the vertical sections design to estimate surface area is described in detail and applied to estimate the area of the pial surface and of the boundary between cortex and underlying white matter (i.e. subcortical surface area). For completeness, cortical volume and mean cortical thickness are also estimated. The aforementioned surfaces were triangulated in 3D with the aid of FreeSurfer software, which provided accurate surface area measures that served as gold standards. Furthermore, a software was developed to produce digitized trace curves of the triangulated target surfaces automatically from virtual sections. From such traces, a new method (called the ‘lambda method’) is presented to estimate surface area automatically. In addition, with the new software, intersections could be counted automatically between the relevant surface traces and a cycloid test grid for the classical design. This capability, together with the aforementioned gold standard, enabled us to thoroughly check the performance and the variability of the different estimators by Monte Carlo simulations for studying the human brain. In particular, new methods are offered to split the total error variance into the orientations, sectioning and cycloid components. The latter prediction was hitherto unavailable – one is proposed here and checked by way of simulations on a given set of digitized vertical sections with automatically superimposed cycloid grids of three different sizes. Concrete and detailed recommendations are given to implement the methods.     

Two rapid and simple methods used for the removal of resins from 1.0 micron thick epoxy sections.

Ultrathin epoxy sections are commonly used in electron microscopy. More useful information can often be obtained by examining thicker 0.5--3.0 micron) resin sections under the light microscope. However, there are some histological stains that are unable to penetrate these resins. This short note describes the easy and rapid preparation of two resin-removal solutions which allow subsequent staining of 1.0 micron thick sections without obvious tissue damage and with excellent results using standard histological dyes.     

A method for sectioning and handling frozen sections for scanning electron microscopy

Glutaraldehyde-fixed insects were embedded in Tissue-Tek^R and sectioned on a liquid-nitrogen-cooled stage. The sections were sandwiched between two layers of microscope lens paper and all postsectioning treatments were carried out on this sandwich, including dehydration and critical-point drying. In some cases, the sections were placed on filter membranes and lyophilized. These procedures produced intact specimens which maintained internal morphology as well as inter- and intracellular integrity without expensive or specialized equipment.     

Electron microscopy of semithick sections: Advantages for biomedical research

Many transmission electron microscopes are available which can be used to examine biological material in 0.25–0.50-μm-thick sections. When compared to the traditional thin section, these “semithick” sections possess a number of inherent advantages: They can be screened for content with the phase contrast light microscope, they facilitate many types of studies requiring an analysis of serial sections, and they are frequently the optimum thickness for stereomicroscopy. Structures such as microtubule-associated components, as well as structural relationships between cellular constituents, may also be clearly visible in semithick sections which are not visible, or go unnoticed, in thin sections. Together these advantages enable an investigator to obtain a more complete three-dimensional picture of a cell or cell component in a significantly (i.e., up to 90%) shorter period of time than would be required if thin sections were used. Semithick sections may, therefore, make a study feasible which is not approachable, or which is approachable only with great difficulty, by conventional thin sectioning techniques.     

Three-dimensional analysis of neuronal geometry using HVEM

There is a need for an electron microscopic method for visualization of selectively stained neurons and neuronal processes with higher resolution than can be obtained with the light microscope, but using thick sections that allow visualization of the three-dimensional structure of the neuron. Such a method is required for measurement of the geometry of neurons, and this information is needed to test theoretical predictions on the way in which electrical signals of synaptic origin are processed by the cells. The high voltage electron microscope (HVEM) is well suited to this application, because of its high resolution and ability to form images of thick sections. Use of this instrument requires development of selective stains that can produce diffuse cytoplasmic staining of specific cells or cell populations on the basis of their functional properties. Several such methods currently being employed for light microscopic work can be used directly in the high voltage electron microscope or can be made useful by relatively minor alterations. These include intracellular staining with horseradish peroxidase, axonal tracing with Phaseolus vulgaris leukoagglutinin (PHA-L), and immunocytochemical staining for specific cell markers known to stain the cytoplasm of certain cell populations. Cells stained intracellularly by microinjection of horseradish peroxidase during physiological recording experiments may be stained in thick (ca. 50 μm) sections cut on a vibratome or similar instrument and stained in the standard way, using methods designed for light microscopy. The sections are then postfixed in osmium tetroxide and embedded in epoxy plastic. Sections cut from these blocks at thicknesses of from 1 to 5 μm using a dry glass knife may be examined directly in the HVEM with no further staining. This produces a very clear image of the cell on a relatively unstained background. This method provides more than adequate resolution of the boundary of the neuron, allowing measurement of neuronal processes to better than 10-nm precision. Similar results are obtained when the same method is applied to axonal tracing using PHA-L. In this case, the exogenously applied marker is used to label a small population of nearby neurons and to trace their connections with other cells at a distance. The lectin is detected by immunocytochemistry, but the selective contrast of the image is adjustable because the concentration of antigen in the cell is largely controlled by the experimenter. The lectin is distributed diffusely in the cytoplasm in a pattern identical to that of intracellular staining, so like intracellular staining, it reveals the overall shape of the cell. Immunocytochemical labelling using endogenous antigens known to be distributed in the cytoplasm of specific neurons produced inadequate control of selective contrast when prepared in this manner. Instead, 1–10μm sections cut from blocks of nervous tissue were embedded in polyethylene glycol, stained using a combedded in polyethylene glycol, stained using a combination of immunocytochemistry and histochemical intensification methods, and embedded in plastic on the grid. This method, which is also suited for staining with poorly penetrating markers such as colloidal gold, may also prove useful in a variety of other situations requiring the intensification of selective contrast.     

The saucor, a new stereological tool for analysing the spatial distributions of cells, exemplified by human neocortical neurons and glial cells

The 3D spatial arrangement of particles or cells, for example glial cells, with respect to other particles or cells, for example neurons, can be characterized by the radial number density function, which expresses the number density of so-called 'secondary' particles as a function of their distance to a 'primary' particle. The present paper introduces a new stereological method, the saucor, for estimating the radial number density using thick isotropic uniform random or vertical uniform random sections. In the first estimation step, primary particles are registered in a disector. Subsequently, smaller counting windows are drawn with random orientation around every primary particle, and the positions of all secondary particles within the windows are recorded. The shape of the counting windows is designed such that a large portion of the volume close to the primary particle is examined and a smaller portion of the volume as the distance to the primary object increases. The experimenter can determine the relation between these volumina as a function of the distance by adjusting the parameters of the window graph, and thus reach a good balance between workload and obtained information. Estimation formulae based on the Horvitz-Thompson theorem are derived for both isotropic uniform random and vertical uniform random designs. The method is illustrated with an example where the radial number density of neurons and glial cells around neurons in the human neocortex is estimated using thick vertical sections for light microscopy. The results indicate that the glial cells are clustered around the neurons and the neurons have a tendency towards repulsion from each other.     

Automated identification of neurons and their locations

Individual locations of many neuronal cell bodies (>10⁴) are needed to enable statistically significant measurements of spatial organization within the brain such as nearest‐neighbour and microcolumnarity measurements. In this paper, we introduce an Automated Neuron Recognition Algorithm (ANRA) which obtains the (x, y) location of individual neurons within digitized images of Nissl‐stained, 30 μm thick, frozen sections of the cerebral cortex of the Rhesus monkey. Identification of neurons within such Nissl‐stained sections is inherently difficult due to the variability in neuron staining, the overlap of neurons, the presence of partial or damaged neurons at tissue surfaces, and the presence of non‐neuron objects, such as glial cells, blood vessels, and random artefacts. To overcome these challenges and identify neurons, ANRA applies a combination of image segmentation and machine learning. The steps involve active contour segmentation to find outlines of potential neuron cell bodies followed by artificial neural network training using the segmentation properties (size, optical density, gyration, etc.) to distinguish between neuron and non‐neuron segmentations. ANRA positively identifies 86 ± 5% neurons with 15 ± 8% error (mean ± SD) on a wide range of Nissl‐stained images, whereas semi‐automatic methods obtain 80 ± 7%/17 ± 12%. A further advantage of ANRA is that it affords an unlimited increase in speed from semi‐automatic methods, and is computationally efficient, with the ability to recognize ～100 neurons per minute using a standard personal computer. ANRA is amenable to analysis of huge photo‐montages of Nissl‐stained tissue, thereby opening the door to fast, efficient and quantitative analysis of vast stores of archival material that exist in laboratories and research collections around the world.     

Celloidin–wax sandwich microtomy: a novel and rapid method for producing serial semithin sections

A method is described that allows rapid and reliable serial sectioning down to thicknesses of 1 μm. The tissue is first embedded in celloidin and then in wax and trimmed so that the block is sandwiched between two layers of wax. This combines the virtues of both media. The celloidin gives greater support to tissue than wax and enables the cutting of semithin sections. The wax allows ribbons of serial sections to be produced as in conventional wax microtomy. This makes it easy to produce serial semithin sections as a matter of routine.     

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.     

An easy brain thick-section-clearing protocol to observe antigens in the brains of neurological disease mouse models by conventional epifluorescence and confocal laser scanning microscopy

We developed a brain thick-section-clearing (BTS-C) procedure to observe coronal sections of the entire mouse brain with conventional epifluorescence microscopy (EFM). After clearing with the BTS procedure, the white light transmittance of the 1-mm-thick mouse brain sections was more than 96%. The distribution patterns of amyloid plaques or α-synuclein in 1-mm-thick brain sections of five different mouse strains cleared with this procedure were easily observed by EFM. In addition, the detailed distribution of antigens at higher magnifications was revealed by observing the same slides with a confocal laser scanning microscope (CLSM). In conclusion, we have shown that the BTS protocol can replace laborious and time-consuming semi-thin-sectioning procedures, and the cleared 1-mm-thick sections can be examined by EFM and CLSM to elucidate the distribution patterns of antigens.     

Improvement in the resolution of three-dimensional data sets collected using optical serial sectioning

In this paper an approach for improving the quality of 3-D microscopic images obtained through optical serial sectioning is described and implemented. A serially sectioned image is composed of a sequence of 2-D images obtained by incrementing the focusing plane of the microscope through the specimen of interest; ideally, the image obtained at each focusing plane should be in focus, and should contain information lying only within that plane. In practice, however, the images obtained contain redundant information from neighbouring focusing planes and are blurred by a three-dimensional low-pass distortion. These degradations are a consequence of the limited aperture of any optical system; using principles of geometric optics and allowing for the passage of light through the specimen, we are able to demonstrate that the microscope distortion can be described as a linear system, if the absorption of the specimen is assumed to be linear and non-diffractive. The transfer function of the microscope is found to zero a biconic region of 3-D spatial frequencies orientated along the optical axis; a closed-form expression is derived for the low-pass transfer function of the microscope outside the region of missing frequencies. The planar resolution of the serial sections can be greatly improved by convolving the image obtained with the inverse of the low-pass distortion function, although the missing cone of frequencies is not recoverable. The reconstruction technique is demonstrated using both simulated images, to demonstrate more clearly the effects of the distortion and the accuracy of the subsequent reconstruction, and actual experiments with a pollen grain and a stained preparation of human cerebellum tissue.