Quantitative Histology Using Confocal Microscopy: Implementation of Unbiased Stereology Procedures

Direct, two-dimensional counting or measuring of cells as they appear in histological sections is subject to a number of artifacts that can lead to erroneous conclusions about changes in cellular populations. Numerous correction procedures devised to compensate for these artifacts are collectively termed model-based stereology due to their reliance on a model of cell geometry for correction formulas. These corrections are valid only to the degree that the geometric model reflects cellular morphology. In addition, there are requirements for population homogeneity that are often not met in biological material. The development of design-based stereology provides a way to directly count or measure cells in three dimensions, avoiding errors (biases) and the need for assumptions regarding cell size, shape, and orientation to be validated. On this basis, these procedures are described as unbiased stereology. The recent commercial availability of semiautomated stereology systems has substantially reduced the effort and experimenter error (bias) associated with the use of design-based stereology. The optical resolution of confocal microscopy and the ability to collect registered series of focal planes is ideally suited for the three-dimensional sampling of design-based stereology. Unfortunately, stereological procedures are not available in any confocal microscope software and it is up to the user to implement these procedures. Strategies and illustrations of approaches to implementing stereological procedures on a confocal microscope are presented. Where possible, particular design issues are discussed and solutions suggested. With user requests, future generations of confocal software may integrate collection of confocal images with the implementation of design-based stereology.     

Stereological evaluation of the soleus muscle isografted into fast extensor digitorum longus (EDL) muscle in rats with different thyroid status

The 2-D stereology can be used advantageously in the case of muscle cross sections stained by routine histochemical and immunocytochemical methods, such as mATPase reaction, when the quality of the image is often not sufficient for using image analysis techniques without considerable individual intervention. Other advantages of stereological methods in muscle morphometry are that measurements are made directly on specimens under the microscope and in their simplest arrangement they do not require sophisticated and expensive technical equipment. Furthermore, unbiased results are obtained, no segmentation and edge effect problems arise and the quantity of work invested in stereological estimation is reasonable. Therefore, we have used the stereological methods as our standard technique for assessment of fibre type composition in regenerated soleus muscles grafted from 21- to 28-day-old rats into fast EDL muscles of adult inbred recipients with different plasma levels of thyroid hormones.     

Recent Stereological Methods for the Measurement of Leaf Anatomical Characteristics: Estimation of Volume Density, Volume and Surface Area

Recent design-based stereological methods for the measurementof the proportion of mesophyll in the leaf, the proportion ofintercellular spaces in the mesophyll, the leaf volume, themesophyll volume, and the exposed leaf surface area of mesophyllare described in an easy-to-follow manner. Two leaf types areconsidered: grass leaf and bifacial leaf. The presented methodsare discussed and compared with alternative methods. Key words: Cavalieri's principle, leaf, mesophyll, stereology, vertical sections     

Recent stereological methods for measuring leaf anatomical characteristics: estimation of the number and sizes of stomata and mesophyll cells

Recent design-based stereological methods for measuring stomatalfrequency, mean epidermal cell length, number of mesophyll cellsand mean cell volume are described in an easy-to-follow manner.Two leaf types are considered: grass leaf and bifacial leaf.The presented methods are discussed and compared with alternativemethods. Key words: Disector, leaf, mesophyll, stereology, stomata     

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission election microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.     

Comparison of several digital and stereological methods for estimating surface area and volume of cells studied by confocal microscopy.

BACKGROUND: The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS: Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS: It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS: The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.     

Stereology techniques in radiation biology

Clinicians involved in conventional radiation therapy are very concerned about the dose-response relationships of normal tissues. Before proceeding to new clinical protocols, radiation biologists involved with conformal proton therapy believe it is necessary to quantify the dose response and tolerance of the organs and tissues that will be irradiated. An important focus is on the vasculature. This presentation reviews the methodology and format of using confocal microscopy and stereological methods to quantify tissue parameters, cell number, tissue volume and surface area, and vessel length using the microvasculature as a model tissue. Stereological methods and their concepts are illustrated using an ongoing study of the dose response of the microvessels in proton-irradiated hemibrain. Methods for estimating the volume of the brain and the brain cortex, the total number of endothelial cells in cortical microvessels, the length of cortical microvessels, and the total surface area of cortical microvessel walls are presented step by step in a way understandable for readers with little mathematical background. It is shown that stereological techniques, based on a sound theoretical basis, are powerful and reliable and have been used successfully.     

Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.     

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections², generates reliable structural data for biological features in the mammalian brain³. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space⁴. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.Open in a separate windowClick here to view.^{(58M, flv)}     

Towards a computer aided diagnosis system dedicated to virtual microscopy based on stereology sampling and diffusion maps

An original strategy is presented, combining stereological sampling methods based on test grids and data reduction methods based on diffusion maps, in order to build a knowledge image database with no bias introduced by a subjective choice of exploration areas. The practical application of the exposed methodology concerns virtual slides of breast tumors.     

daime, a novel image analysis program for microbial ecology and biofilm research

Combinations of microscopy and molecular techniques to detect, identify and characterize microorganisms in environmental and medical samples are widely used in microbial ecology and biofilm research. The scope of these methods, which include fluorescence in situ hybridization (FISH) with rRNA-targeted probes, is extended by digital image analysis routines that extract from micrographs important quantitative data. Here we introduce daime (digital image analysis in microbial ecology), a new computer program integrating 2-D and 3-D image analysis and visualization functionality, which has previously not been available in a single open-source software package. For example, daime automatically finds 2-D and 3-D objects in images and confocal image stacks, and offers special functions for quantifying microbial populations and evaluating new FISH probes. A novel feature is the quantification of spatial localization patterns of microorganisms in complex samples like biofilms. In combination with '3D-FISH', which preserves the 3-D structure of samples, this stereological technique was applied in a proof of principle experiment on activated sludge and provided quantitative evidence that functionally linked ammonia and nitrite oxidizers cluster together in their habitat. This image analysis method complements recent molecular techniques for analysing structure-function relationships in microbial communities and will help to characterize symbiotic interactions among microorganisms.     

On the Stereological Estimation of Numerical Density of Particle Systems by an Object Counting Method

The determination of numerical density Nv of particles is one of the most complex problems in stereology. This paper presents a method for solving it, consisting only of counting of intersections between particles and thick slices of some different thicknesses. The essential stereological parameters Nv and mean caliper diameter D of the particles are determined by means of a regression line. The basic formula is a simple consequence of general principles for deriving stereological formulae which is described in this paper. The object counting method is applicable if the particles are convex and if the whole particle system can be described by a stationary isotropic ergodic marked point process. A practical example (liver cell nuclei of rats) demonstrates the application of the method.     

Application of immunohistochemistry in stereology for quantitative assessment of neural cell populations illustrated in the göttingen minipig

Background

Stereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification.

Methodology/Principal Findings

We propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Göttingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number.

Conclusion/Significance

The concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.     

Stereologic analysis of vacuolization of the T-system of frog muscle fibers, detected using confocal fluorescence microscopy

The confocal fluorescence microscopy has been used for quantitative evaluation of the T-system reversible vacuoles produced by efflux of 80-120 mM glycerol from frog skeletal muscle fibers. The fibers were stained by membrane probe RH414 and by water-soluble dye fluorescein dextran that marks the vacuolar lumen. Using morphometrical and stereological methods the volume and surface densities of vacuoles were measured on single optical sections and Z-series during a 30 min glycerol efflux. Various methods of measurements (three-dimensional reconstruction of vacuoles, computer morphometry, point counting method) applied to the same Z-series provide similar results. The vacuolar membranes stained by RH414 look like bright rings 0.3-0.4 micron in width. It is concluded that the real position of vacuolar membrane corresponds to the middle of the vacuolar envelope. The measurements of the external dimensions of the envelope overestimate the stereological parameters up to 50%. The volume density of vacuoles reaches 10% within 20-30 min of glycerol efflux. It means that the volume of the T-system may increase by 25-30 times compared to the control value (0.3-0.4%). The surface density of vacuoles during reversible vacuolation is equal to 0.20-0.35 micron-1 and does not exceed the surface density of normal T-system. The sufficiency of membrane material for the T-system reversible vacuolation is discussed in addition to the role of geometrical factor in this phenomenon.     

Characterization of the capillary network in skeletal muscles from 3D data

In this review we present immunohistochemical methods for visualization of capillaries and muscle fibres in thick muscle sections. Special attention is paid to the procedures that preserve good morphology. Applying confocal microscopy and virtual 3D stereological grids, or tracing of capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to a muscle fibre per fibre length, fibre surface area or fibre volume can be evaluated by an unbiased approach. Moreover, 3D models of capillaries and muscle fibres can be produced. Comparison of the developed methods with counting capillary profiles from 2D sections is discussed and the reader is warned that counting capillary profiles from 2D sections can underestimate the capillary length by as much as 75 percent. Application of the described 3D methodology is illustrated by the anatomical remodelling of capillarity during acute denervation and early reinnervation in the rat soleus and extensor digitorum longus muscles.     

Morphometric and Stereologic Analysis of Chlorella vulgaris Under Heterotrophic Growth Conditions

A computer-supported determination of stereological parameterswas used to study the possible ultrastructural changes of Chlorellavulgaris UAM 101 under photolithotrophic, mixotrophic and photoheterotrophicconditions of growth. Data recording was carried out througha semi-automatic digitizing image analysis system instead ofthe current method of superimposition of an array of short lines. Glucose promoted drastic physiological changes [Martínezand Orús, 1991. Plant Physiology (in press)], which stronglyaffected the size of the cells and volume densities of storagematerials. However, volumetric ratios of the mitochondrion orchloroplast active fraction were not affected by the presenceof glucose, probably indicating that these ratios are characteristicof each species. Cell wall ultrastructure was also analysed and the presenceof sporopollenin demonstrated, in contrast to the thin and sporopollenin-lackingcell wall generally described for Chlorella vulgaris species. Chlorella vulgaris, photoautotrophic, mixotrophic, photoheterotrophic, image analysis, stereology     

Visualisation and stereological assessment of blood and lymphatic vessels

The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.     

Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study

The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.