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%.     

x-y-Recording in transmission electron microscopy. A versatile and inexpensive interface to personal computers with application to stereology

An interface for IBM XT/AT-compatible computers is described which has been designed to read the actual specimen stage position of electron microscopes. The complete system consists of (i) optical incremental encoders attached to the x- and y-stage drivers of the microscope, (ii) two keypads for operator input, (iii) an interface card fitted to the bus of the personal computer, (iv) a standard configuration IBM XT (or compatible) personal computer optionally equipped with a (v) HP Graphic Language controllable colour plotter. The small size of the encoders and their connection to the stage drivers by simple ribbed belts allows an easy adaptation of the system to most electron microscopes. Operation of the interface card itself is supported by any high-level language available for personal computers. By the modular concept of these languages, the system can be customized to various applications, and no computer expertise is needed for actual operation. The present configuration offers an inexpensive attachment, which covers a wide range of applications from a simple notebook to high-resolution (200-nm) mapping of tissue. Since section coordinates can be processed in real-time, stereological estimations can be derived directly “on microscope”. This is exemplified by an application in which particle numbers were determined by the disector method.     

Computer-assisted stereology: point fraction of lung parenchyma and alveolar surface density in fetal and newborn sheep

The aim of this study was to determine the accuracy of a new computer-assisted stereological technique in obtaining structural information of the lung. We compared the point fraction of lung parenchyma (Pp) and alveolar surface density (Sv) obtained by established manual point/intercept counting methods and compared them with those obtained using a computer-assisted method. Lung tissues obtained from normally grown fetal sheep (n = 6) and from newborn lambs with severe lung hypoplasia (n = 5) were inflation fixed via the trachea and processed for light microscopy. In verification-of-technique experiments, Pp and Sv correlated well with known values. There was a significant linear correlation between manual and computer-assisted stereological measurements for values of Pp (r2 = 0.92) and Sv (r2 = 0.98). Our data lead us to believe that the computer-assisted stereological technique described in this study provides accurate estimates of Pp and Sv and hence may be a valuable tool for evaluating the effects of factors upon structural development of the lung.     

Characterization of nickel silicides using EELS-based methods

We present an unbiased estimator of the total number of alveolar structures distal to the transition from a bronchiole to an alveolar duct system (‘ventilatory units’, VUs). In species without respiratory bronchioles, including mice, the number of VUs is equivalent to the number of acini. The acinus is a functional unit of gas exchange, defined as a parenchymal unit distal to a terminal bronchiole in which all airways contain alveoli and thus participate in gas exchange. The estimator combines two different estimators of the number of VUs: (1) an estimator derived from the Euler number of all the openings of the bronchial tree and (2) an estimator derived from direct counts of topological changes occurring at bronchiole‐alveolar duct junctions. Combining the two estimators eliminates the requirement to be able to identify even vanishingly small pieces of bronchial tissue in physical disectors. We implemented the fractionator estimator in five adult mice lungs using physical fractionators with varying but known sampling fractions (Horvitz–Thompson estimator). We obtained total values of about 4200 VUs (CV = 0.05) in 21‐day‐old and 4480 (CV = 0.06) in 69‐day‐old animals. Being fractionator estimates, these total numbers are independent of shrinkage. The densities of VUs per unit volume of tissue (values corrected for tissue shrinkage) were similar in left and right lungs.     

Application of the disector method in the light microscopic quantification of type II pneumocytes in control and ozone-exposed rats

Alveolar type II cells in control and ozone-exposed rat lungs were counted at the light microscopical level with the ‘disector method’. The type II cells were unequivocally marked by histochemical staining for alkaline phosphatase activity in 2 μm plastic sections. By this counting method, the mean number of type II cells per lung in control rats was of the same magnitude as those reported in the literature, using point counting methods. After exposure of rats to 1.6 mg ozone/m³ for 7 days, a 50% increase in the mean number of type II cells was observed. The use of the disector method at the light microscopical level offers some advantages above a quantification at the electron microscopical level. The procedure is less time-consuming, larger areas can be screened, two parallel countings can be performed in one set of sections and there is no need for an exact knowledge about the diameter of the measured particle.     

The estimation of the Euler-Poincare characteristic from observations on parallel sections

An unbiased estimator of the Euler-Poincaré characteristic (Euler number) of an arbitrary object or a random structure, respectively, is given. The estimator is based on joint observations of pairs of parallel section profiles. Thus the present paper extends the use of Sterio's disector from counting particles to determining the Euler number for a wide class of probes. The correctness of the given formulae is proved with mathematical strictness. Furthermore, the feasibility of the method is illustrated by an example from materials sciences.     

A brief introduction to computer-intensive methods, with a view towards applications in spatial statistics and stereology

Computer-intensive methods may be defined as data analytical procedures involving a huge number of highly repetitive computations. We mention resampling methods with replacement (bootstrap methods), resampling methods without replacement (randomization tests) and simulation methods. The resampling methods are based on simple and robust principles and are largely free from distributional assumptions. Bootstrap methods may be used to compute confidence intervals for a scalar model parameter and for summary statistics from replicated planar point patterns, and for significance tests. For some simple models of planar point processes, point patterns can be simulated by elementary Monte Carlo methods. The simulation of models with more complex interaction properties usually requires more advanced computing methods. In this context, we mention simulation of Gibbs processes with Markov chain Monte Carlo methods using the Metropolis-Hastings algorithm. An alternative to simulations on the basis of a parametric model consists of stochastic reconstruction methods. The basic ideas behind the methods are briefly reviewed and illustrated by simple worked examples in order to encourage novices in the field to use computer-intensive methods.     

Recent applications of the new stereology have thrown fresh light on how the human placenta grows and develops its form

The availability of design-based stereological methods has made it possible to readdress certain key and contentious issues in placental growth and morphogenesis. Three particular questions are: (i) does the population of cytotrophoblast cells decline during gestation?, (ii) is placental growth biphasic or monophasic? and (iii) what are the consequences for intervillous porosity of the elaboration of terminal villi? These questions cannot be answered definitively without recourse to the new stereology. Applying the disector to estimate nuclear number and star volume to assess pore size, recent studies have helped to resolve these issues. Their findings are reviewed. Nuclei were counted in the trophoblastic epithelium, stroma and vascular endothelium of placental villi. It was found that growth is monophasic and proliferative. All types of nuclei increased in number throughout gestation and this included cytotrophoblast. Trophoblast grows by the continuous recruitment of new proliferative units of uniform mean volume. The so-called ‘loss’ of cytotrophoblast cells is a misinterpretation of what is seen on microscopical sections and is attributable to disproportionate growth in villous surface area. Cells simply become more widely dispersed. Elaboration of finer terminal branches on villous trees leads to a decline in the star volumes of villi and intervillous pores. Some of the functional implications of these findings are discussed.     

Vertical LM sectioning and parallel CT scanning designs for stereology: application to human lung

Practical, unbiased stereological methods are described to estimate lung volume and external surface area, and total volume and surface area of relatively large and anisotropic structures (bronchi and arteries) inside the lung. The volume of each of five lung strata was estimated first by fluid displacement and then by computed tomography (CT) using Cavalieri's method; the reliability of CT was assessed through a calibration procedure, and image thresholding criteria for an accurate volume estimation using CT were established. The parallel, perfectly registered CT section images were also used to estimate the external surface area of each stratum by the spatial grid method. Unbiased estimation of internal surface areas in lung is a long-standing problem: since the structures are large and essentially void, large sections are needed; to facilitate identification, thin sections have to be used for light microscopy, and since such structures are anisotropic, the sections should be vertical. A practical stereological design is demonstrated here on an infant lung, which fulfils all these requirements. This study illustrates the potential of using unbiased stereology to characterize infant pulmonary hypoplasia.     

浅谈云状判断中易出现的几种误记

在云的观测中,通过对比较易出现误记的积雨云与雨层云、雨层云和蔽光层积云、复高积云和积雨云、卷层云和高层云、高云和低云的表现特征进行分析,从而提高对云的观测水平。     

A histological and immunohistochemical study on the parabronchial epithelium of the domestic fowl's (Gallus gallus domesticus) lung with special reference to its scanning and transmission electron microscopic characteristics

The current study was designed to give complete histo-and immunohistochemical features of the parabronchial epithelium of domestic fowl's (Gallus gallus domesticus) lung with special reference to Scanning electron microscope (SEM) and mean transmission electron microscope (TEM) features. The lung exhibited variable-sized atrial openings encircled by exchange tissue zones. The parabronchial atrial chambers appeared as ovoid and polygonal-shaped that separated by the well-developed interatrial septum. The deep atrial lumens had blood vessels pierced by openings that represent the infundibula. The parabronchial blood capillaries meshwork was branched and exhibited ovoid-shaped air capillaries with numerous extravasated blood vessels. By TEM, there were several air capillaries and groups of squamous and endothelial respiratory cells and the squamous cells had oval nucleus with evenly distributed chromatin. The endothelial respiratory cells had few microvilli on their free surfaces. The parabronchial tubes opened into a group of widened atria that had smooth muscle bundles at the interatrial septa. The atrial chambers led to narrow infundibula. Moreover, the lining epithelium of parabronchi, atria, infundibula, and air capillaries was formed by simple squamous epithelium. Air capillary walls were lined by two types of respiratory cells (Types-I and II). Collagen fibers were concentrated within the tunica externa layers of the parabronchial blood vessels as well as, they were observed in CT interparabronchial septa. Immunohistochemically, the elastin immunoreactivity was detected around the parabronchial blood vessels, at the base of each parabronchial atria, and in the area encircling the alveolar-capillary walls. Our work concluded that there are a relation between the fowl's lifestyle and the surrounding environmental conditions.     

浅谈MAG焊和埋弧焊在箱式井架的应用

从箱式井架的结构形状和焊缝接头形式出发,针对MAG焊和埋弧焊的特点进行了论述,生产结果证明,通过两种优质、高效方法的运用,使焊接质量得到了保障,生产效率得到很大提高.     

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT₁) and Endothelin 1 type A (ET_A) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT₁ and ET_A receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT₁ receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ET_A receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.