Optical disector counting in cryosections and vibratome sections underestimates particle numbers: effects of tissue quality

Optical disector counting is currently applied most often to cryosections, followed in frequency by resin-embedded tissues, paraffin, and vibratome sections. The preservation quality of these embedding options differs considerably; yet, the effect of tissue morphology on numerical estimates is unknown. We tested whether different embedding media significantly influence numerical estimates in optical disector counting, using the previously calibrated trochlear motor nucleus of hatchling chickens. Animals were perfusion-fixed with paraformaldehyde (PFA) only or in addition with glutaraldehyde (GA), or by Methacarn immersion fixation. Brains were prepared for paraffin, cryo-, vibratome- or celloidin sectioning. Complete penetration of the thionin stain was verified by z-axis analysis. Neuronal nuclei were counted using an unbiased counting rule, numbers were averaged for each group and compared by ANOVA. In paraffin sections, 906 +/- 12 (SEM) neurons were counted, similar to previous calibrated data series, and results obtained from fixation with Methacarn or PFA were statistically indistinguishable. In celloidin sections, 912 +/- 28 neurons were counted-not statistically different from paraffin. In cryosections, 812 +/- 12 neurons were counted (underestimate of 10.4%) when fixed with PFA only, but 867 +/- 17 neurons were counted when fixed with PFA and GA. Vibratome sections had the most serious aberration with 729 +/- 31 neurons-a deficit of 20%. Thus, our analysis shows that PFA-fixed cryosections and vibratome sections result in a substantial numerical deficit. The addition of GA to the PFA fixative significantly improved counts in cryosections. These results may explain, in part, the significant numerical differences reported from different labs and should help investigators select optimal conditions for quantitative morphological studies.     

The revolution of counting "tops": two decades of the disector principle in morphological research

Twenty years have passed since the publication of the seminal paper enunciating the disector principle by an author using the pseudonym D.C. Sterio. During this time, methods based on the revolutionary principle of counting "tops" have become progressively better known and have been included in several commercially available systems for quantitative morphology. Analysis of the number of published studies citing Sterio's paper on the ISI Web of Knowledge database showed that its scientific "impact factor" has almost continuously risen since its publication, indicating the growing knowledge about disector-based methods in the various scientific fields where morphological quantification is required. This report briefly reviews the first two decades of disector use, pointing to its advantages as well as to shortcomings that have recently been addressed in critical papers and have given rise to a lively debate on the role of counting tops in quantitative morphology today.     

Unbiased and efficient estimation of the total number of terminal bronchiolar duct endings in lung: a modified physical disector

A novel modification of the physical disector is described which was used to estimate the total number of terminal bronchiolar duct endings (TBDEs) in human infant lung. TBDEs are closed three-dimensional space curves of complex shape that are inherently difficult to count from histological sections. However, careful consideration of the microanatomy of the terminal duct endings provides us with the opportunity to define a very simple and unbiased counting rule. To apply the rule in practice we also need to determine a suitable disector height. Owing to the complex shape of the TBDE we had no prior knowledge of what disector height would be suitable for counting the TBDE structures. Exhaustive serial sectioning of complete TBDE structures was carried out and showed that any disector height under 90 μm would give unbiased counts. A further empirical study was then undertaken to determine the most efficient disector height. This was found to be 50 μm.
The total number of TBDEs in the upper lobe of the right lung of six human infants aged between 13 and 25 weeks was also estimated. The estimates of numerical density obtained with our modification of the physical disector were multiplied by estimates of lung lobe volume obtained using Cavalieri's Principle. The total number of TBDEs in the lobes ranged from 15 323 to 57 768, with a mean of 40 306. The average coefficient of error of the number estimates was 19%, which was deemed precise enough given the biological coefficient of variation between TBDE number of 36%.     

Stereological estimation using vertical sections in a complex tissue

A method designed for stereological estimation in a very complex tissue using vertical sections is presented. In some tissues, the random rotation of the tissue for vertical sections may obscure recognition of the anatomical structures of interest. The present method overcomes this problem by generating sections with both a particular orientation, 'mapping sections', and ordinary random vertical sections usable for the required observations. A map describing the positions of the vertical sections is produced to make the complex reference space recognizable. The method is illustrated by estimating the number and size of neurones in the dorsal raphe nucleus of the human brainstem with its dense packing of roughly 100 nuclei within a volume less than 50 cm³.     

Estimation of the section thickness and optical disector height with a simple calibration method

The distance between the upper and lower surfaces of a section (i.e. the section thickness) can be measured with a microcator or a shaft encoder. In the present report, an alternative simple method is described for estimating the section thickness where such equipment is not available. The basic principle of the method is based on a calibration method already described in the literature. The main difference is that it enables one to make more precise measurements. Provided that the calibration and measurements are made properly, this method can be used in estimating the section thickness, optical disector heights, and in particular in the determination of the thickness sampling fraction for the optical fractionator.     

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.     

Automated cell counting in tissue sections: a new approach by ‘multiple grey-level analysis‘

A system is described for the automated quantitation in tissue sections of the follicular and stromal cell populations of the thyroid gland, using a computer-linked TV image analyser. The paper illustrates how the problems of variable staining intensity and clustering of images (nuclei) may be overcome by a novel approach, in which multiple analyses of each field are made at increasing grey-level thresholds, and then ‘synthesized’ by computer to give a composite image. Discrimination between cell (nuclear) types was by a single minimum width criterion. The results were highly reproducible and correlated well with counts obtained by a comparable manual method.     

Non‐invasive,label‐free cell counting and quantitative analysis of adherent cells using digital holography

Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label‐free and completely non‐invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor™ (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non‐invasive automatic cell counting as precisely as conventional manual cell counting     

The total number of neurons in the human neocortex unbiasedly estimated using optical disectors

An efficient method is presented for obtaining, in under 4h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of ～ 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7-mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80-year-old men was found to be 13·7 × 10⁹ with an inter-individual coefficient of variation of 12%.     

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.     

Estimation of subcellular organelle volume from ultrathin sections through centrioles with a discretized version of the vertical rotator

A method for the fast and efficient estimation of the volume (but not surface area) of subcellular organelles is presented. It consists of a rotator/coaxial-section approach based on the Pappus theorem and represents a discretized version of the vertical rotator where, instead of measuring intercept lengths, the points in distance classes are counted. Centrioles serve as a unique reference 'double-point' with constant size allowing unbiased cell selection from the whole population with equal probability and without the disector application. The sandwich-like method of sample preparation allows comparison of control and experimental cases with the same errors caused by overlapping and overprojection. Test experiments demonstrated that the vertical discretized rotator was an efficient and precise tool for the estimation of volume and that a few independent sections of unknown thickness were sufficient for the quantification of one experimental point.     

Serial section reconstruction using a computer graphics system: Applications to intracellular structures in yeast cells and to the periodontal structure of dogs' teeth

A computer graphics system for reconstruction from serial section micrographs was applied to intracellular details of a yeast target cell (Saccharomyces cerevisiae cell) induced by the α factor mating pheromone and was also applied to a periodontal structure of a dog tooth moved orthodontically. In the former, intracellular organelles and a distribution of vesicles could be clearly observed through the cell membrane using the transparent display method in which the smoothing of the reconstructed outer cell membrane surface by computer processing was applied to the transparent display. In the latter case, by cutting through a reconstructed dog tooth and its periodontal tissues, labiolingual and mesiodistal cut surfaces of the tooth and of adjacent alveolar bone could be observed with fine details (232 sections were used).     

Measuring the surface area of a cell by the method of the spatial grid with a CSLM—a demonstration

The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.     

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.     

Development of in situ system to monitor the machining process using a piezo load cell

The measurement of cutting force is one of the most frequently used techniques for monitoring machining processes. Its widespread application ranges from tool condition identification, feedback control and cutting system design to process optimization.This paper suggests another system for measuring cutting force in milling processes. Generally, tool dynamometers are taken into account for the most appropriate cutting force measuring tool in the analysis of a cutting mechanism. However, high prices and limited working space make in situ systems difficult for a controllable milling process. Although an alternative suggestion is to use an AC current from a servomotor, it is unsuitable for cutting force monitoring because of a small upper frequency limit and noise.The suggested cutting force measuring system is composed of two piezo load cells placed between the moving table bracket and the nut flange of the ball screw. It has many advantages, such as lower cost and a wider measurement range than the tool dynamometer, over using the built-in feeding system and the low-cost piezo load cell for applying a conventional machining center.This paper focuses on the performance test of a newly developed measuring system. By comparing the cutting force between the tool dynamometer and the system developed from a series of end milling experiments, the accuracy of the cutting force measurement system was verified. Linearity, transverse sensitivity and the upper frequency limit of the system were verified by experiment.     

Automatic control of mechanical forces acting on cell biomembranes using a vision-guided microrobotic system in computer microscopy

A prototype for automatic control of mechanical forces acting on cell biomembranes is proposed in this paper. This prototype consists of vision-guided position control of the holder and micro-force sensor, automatic mechanical property characterization of cell biomembranes and automatic control of mechanical forces acting on cell biomembranes. A template-free calibration method and autofocusing of multiple objects are introduced in the vision-guided position control to minimize external biological contamination and position the cell, holder and micro-force sensor into the same focal plane, respectively. A third-order polynomial modified from biomembrane point-load model describing the relationship between the measured mechanical force and the deformations of biomembranes is proposed. This simplified model is easily identified and inversed to facilitate the automatic control of mechanical forces. Experimental results based on zebrafish embryos demonstrate the feasibility of the proposed prototype.     

Experimental investigation of dynamic responses of a transparent PEM fuel cell to step changes in cell current density with operating temperature

The dynamic responses of a proton exchange membrane fuel cell (PEMFC) are closely related to the novel water management technique used for the efficient operation of automotive PEMFCs. In order to better understand the dynamic water transport during cell transients, this paper presents an experimental investigation of the transient response of a cell under fully humidified conditions. The cell dynamic performance was measured by employing a transparent cell and investigated with visualization images of the water distribution in the flow channels. Furthermore, the effect of the operating temperature on the cell transients was examined. The results show that the cell dynamic behavior for the tested operating temperature (30–50 °C) conditions is mainly governed by water transport characteristics related to cathode flooding. Also, we show that the time needed for the cell to reach steady-state after a current density step increase is retarded due to excessive water accumulation inside the cell at lower operating temperatures. This paper was recommended for publication in revised form by Associate Editor Ohchae Kwon Han-Sang Kim received his B.S. and M.S. degrees from the Department of Mechanical Engineering at Seoul National University in 1989 and 1991, respectively. Since 1991, he had worked for the R & D Center of Hyundai Motor Com-pany for ten years. He then obtained his Ph.D. degree from Seoul Natonal University in 2005. He is currently a BK21 associate professor in the School of Mechanical and Aerospace Engineering at Seoul National University. Kyoungdoug Min received his B.S. and M.S. degrees from the Department of Mechanical Engineering at Seoul National University in 1986 and 1988, respectively. He then obtained his Ph.D. degree from M.I.T. in 1994. He is currently a professor in the School of Mechanical and Aerospace Engineering at Seoul National University.     

Displacement fields using correlation methods as a tool to investigate cell migration in 3D collagen gels

Living cells embedded in a complex extra-cellular matrix migrate in a sophisticated way thanks to adhesions to matrix fibres and contractility. It is important to know what kind of forces are exerted by the cells. Here, we use reflectance confocal microscopy to locate fibres accurately and determine displacement fields. Correlation techniques are used to this aim, coupled with proper digital image processing. Benchmark tests validate the method in the case of shear and stretching motions. Finally, the method is tested successfully for studying cancer cells migrating in collagen gels of different concentration.     

Quantifying the growth of chlamydia suis in cell culture using high‐content microscopy

The porcine pathogen Chlamydia suis is widespread in pig farming. Isolation of Chlamydia suis in cell culture is crucial for the generation and characterization of new isolates. However, isolation of Chlamydia suis strains from field samples is fastidious. Therefore, we exploited high‐content microscopy to quantify the growth of Chlamydia suis strains in different cell lines. We found that the cell line yielding optimal propagation of Chlamydia suis differed among isolates, and we identified cell lines outperforming those routinely used for chlamydial isolation. We conclude that adaptation of the propagation procedure to the origin of the putative field isolate is highly recommended to improve the recovery rate.