Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus

The nucleolus of the human Sertoli cell displays a spontaneous segregation of its components and has only one or 2 large fibrillar centers. The 3-dimensional reconstruction and quantitative analysis of its components was undertaken using a Quantimet 900 image analysis system in order to define the spatial relationships between the dense fibrillar component and the fibrillar center and especially to investigate whether threads of dense fibrillar component exist independently, without being linked to a fibrillar center. Our 3D reconstructions demonstrated that the dense fibrillar threads or sheets were never independent of fibrillar centers. These structures belonged to a continuous network that joined the layer of dense fibrils surrounding the fibrillar center. When the nucleolus contained 2 different-sized fibrillar centers, quantitative analysis showed that there was a proportional relationship between the volume of the dense fibrillar component and the volume of the fibrillar center. These data, compared with those previously obtained by means of autoradiographic techniques, suggest that the rDNA-containing chromatin passes through the fibrillar center and unwinds from there into the dense fibrillar component.     

Image analysis combined with quantitative cytochemistry

Summary This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%. In LEYTAS-2, which is based on LEYTAS-1 studies, the microscope is equipped with a new type of objective, enabling the analysis of microscope fields, which are four times as large as in LEYTAS-1. The image analysis part consists of the Modular Image Analysis Computer (MIAC, Leitz) and for alarm storage an eight-bit grey value processor is used. Comparison with LEYTAS-1 shows that cell selection capacities are similar and that the speed is four times higher.In honour of Prof. P. van Duijn     

High-resolution 3D quantitative analysis of caveolar ultrastructure and caveola-cytoskeleton interactions

Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure–function relationships in three-dimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.     

The quantitative ultrastructure of the pea shoot apex in relation to leaf initiation

Summary The ultrastructure of the pea shoot apical meristem was examined quantitatively in longitudinal sections. Photographs were taken at eleven defined positions in the apex, at six developmental stages within a single plastochron. The only change in ultrastructure during the period of a single plastochron was the increase in the proportion of plastids with starch in the central regions of the apex and in the young leaf axils. This increase occurred midway in time between the emergence of successive leaves, at precisely the time that the orientation of growth changes in the region where a new leaf is to emerge. There were quantitative changes in ultrastructure associated with cell differentiation. In the sequence of cell development from the summit of the apex (central zone) to the incipient pith, cell enlargement was accompanied by an increase in the volume of endoplasmic reticulum, dictyosomes, microbodies and vacuoles per cell, an increase in the number of mitochondria, microbodies and vacuoles per cell, and an increase in the volume, but not the number, of plastids per cell. In the sequence of axillary development (before the axillary bud begins to grow) the number of mitochondria per cell decreased as cell volume decreased but the number of plastids per cell remained constant. The number of plastids per cell increased only in the developmental sequence leading to leaf development, in which the number of mitochondria and dictyosomes per cell also increased. There appeared to be no features of ultrastructure, qualitative or quantitative, which could be correlated with the different rates of cell division in different regions of the meristem. The differences in ultrastructure throughout the apex were mainly quantitative and seemed to be associated with cellular differentiation rather than with the plastochronic functioning of the apex during leaf initiation.     

Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis

This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)     

The problem of ascertainment for linkage analysis.

It is generally believed that ascertainment corrections are unnecessary in linkage analysis, provided individuals are selected for study solely on the basis of trait phenotype and not on the basis of marker genotype. The theoretical rationale for this is that standard linkage analytic methods involve conditioning likelihoods on all the trait data, which may be viewed as an application of the ascertainment assumption-free (AAF) method of Ewens and Shute. In this paper, we show that when the observed pedigree structure depends on which relatives within a pedigree happen to have been the probands (proband-dependent, or PD, sampling) conditioning on all the trait data is not a valid application of the AAF method and will result in asymptotically biased estimates of genetic parameters (except under single ascertainment). Furthermore, this result holds even if the recombination fraction R is the only parameter of interest. Since the lod score is proportional to the likelihood of the marker data conditional on all the trait data, this means that when data are obtained under PD sampling the lod score will yield asymptotically biased estimates of R, and that so-called mod scores (i.e., lod scores maximized over both R and parameters theta of the trait distribution) will yield asymptotically biased estimates of R and theta. Furthermore, the problem appears to be intractable, in the sense that it is not possible to formulate the correct likelihood conditional on observed pedigree structure. In this paper we do not investigate the numerical magnitude of the bias, which may be small in many situations. On the other hand, virtually all linkage data sets are collected under PD sampling. Thus, the existence of this bias will be the rule rather than the exception in the usual applications.     

A comparative analysis of the ultrastructure of hydrocarbon-oxidizing micro-organisms.

The ultrastructure of a variety of micro-organisms was compared after growth on hydrocarbon and non-hydrocarbon substrates. Hydrocarbon-grown organisms were characterized by the presence of intracellular electron-transparent inclusions which in many cases appeared membrane-bound. With one exception, non-hydrocarbon-grown organisms did not contain electron-transparent inclusions. Insignificant amounts of poly-beta-hydroxybutyric acid were produced by the hydrocarbon-grown micro-organisms. After growth on hydrocarbons, all the microorganisms had accumulated varying amounts of the respective unmodified hydrocarbon growth substrate.     

The reliability of eye movement quantitative evaluation. I. Saccadic eye movements.

We adopted the estimate of the intraclass coefficient of reliability, R, to evaluate the reliability of saccadic eye movements quantitative analysis. At a one-week interval we recorded refixative saccadic eye movements twice from fifteen healthy subjects by means of the binocular electrooculographic technique. R was computed for the constants and the slopes of the amplitude/duration and the amplitude/peak velocity relationships, for the mean precision values and for the mean latency values adjusted for subject's age. Our data demonstrated that the reliability of saccade parameters is fairly good for the amplitude/peak velocity relationship, good for the precision and very good for the amplitude/duration relationship. Finally, we believe that the normal variability values we obtained can be usefully employed in neurophysiological longitudinal studies not only in normal subjects, but also in pathological condition provided the more reliable parameters and the more adequate strategies to compute normal variability values are chosen.     

Coupling immunoaffinity techniques with MS for quantitative analysis of low-abundance protein biomarkers

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Comparative evaluation of techniques for the harmonic analysis of human motion data

Four mathematical techniques for the estimation of the Fourier coefficients of pseudoperiodic non-exact discrete functions were submitted to comparative evaluation. These techniques were devised within the following basic procedures: (1) numerical harmonic analysis applied either directly or after redistribution of the data points through some interpolation procedure, so that they be evenly spaced in time and exactly fit on cycle period; (2) fitting of the empirical data with an analytical model followed by the calculation of the Fourier integrals of this model. The evaluation was carried out with special reference to the use of these techniques for processing human motion photogrammetric data. The following criteria were used: (1) accuracy of the Fourier coefficient estimates, (2) capability of yielding information about this accuracy, (3) a priori information needed regarding the statistical properties of the experimental error, (4) factors concerning implementation in digital computers. Practical information was obtained for the non-specialist user with regard to the choice of one technique among the several possible in particular circumstances.     

The ultrastructure of the neonatal pig colon.

The neonatal pig colon has several unique structural and developmental features. At birth it has a variable population of epithelial cells which in their arrangement on villus-like protrusions and in their capability for protein uptake into large preformed supranuclear vacuoles closely resemble neonatal ileal cells. Such villus-like protrusions and vacuolated cells are not present in the 2-day-old piglet. On the first day after birth absorptive epithelial cells which lack supranuclear vacuoles transiently accumulate a large number of lipid droplets, each separated from the cytoplasm only by a proteolipid interface. None of the much smaller lipid droplets bounded by a unit membrane of the smooth endoplasmic reticulum and characteristic of normal small intestinal fat uptake were ever seen in these cells. Very few of the large lipid drops remain on the second day after birth. This initial capacity of the colon for protein and lipid uptake never reappears. The pattern of colonic amino acid transport also changes markedly in the first four days of independent life and this may be correlated with the observation that the absorptive cells at birth have microvilli which are twice the length of those on similar cells at and after two days old. These morphological results are discussed in terms of implied functional changes in the neonatal period.     

The reliability of eye movement quantitative evaluation. II. Smooth pursuit eye movements.

We adopted the estimate of the intraclass coefficient of reliability, R, to evaluate the reliability of smooth pursuit eye movement quantitative analysis. At a one-week interval, we recorded twice smooth pursuit eye movements from fifteen healthy subjects by means of the binocular electrooculographic technique. R was computed for the constant and the slope of the target velocity/gain relationships. R values were rated good for the slope and excellent for the constant. Finally, we computed for each parameter the maximum variability value according to two differing methods; on the basis of the within-subjects mean square values, we defined the normal range of biological test-retest variability for the two parameters.     

森林风景功能的计量评价

采用被视频度、林型权重、被视可能性 ,对富阳市富春江两岸风景森林进行计量评价 ,为两岸风景防护林的配置、管理提供正确的决策。通过地形及土地利用图、数值地形模型和视线入射角来确定景观的被视频度 ;引进人口重力模型来表示森林的被视可能性 ;采取一对一比较法判断林型权重 ;利用 Thurstone的比较判断矩阵得到各景观的评价值。通过使用林型权重 ,可判断景区森林布局的合理性 ,而评价值 (Li)的高低可作为森林景观是否需要改造的标志值。研究表明 ,景观的评价值 (Li)受被视可能性 (Gi)和林型权重 (Vi)的影响。景观评价值最高的为杨树苏柳银芽柳条带状混交林、杨树香椿枫香复层林两种木森林类型 ,其次为杨树河柳林带、杨柳枫杨桤木香樟块状混交林、桤木纯林、杨树纸浆林、柳树纸浆林、街道公园绿化带、枫杨纯林、桑树。森林是以小班为单位经营管理的 ,植被、林龄等在同一小班内基本一致 ,森林风景管理也至少要求对每个小班进行评价 ,因此 ,研究时网格大小起码在小班内含有几个网格 ,最适大小为 5 0 m× 5 0 m。     

A quantitative evaluation of cytophotometric DNA analysis in retrospective studies using archival tumor specimens.

Methods developed for the cytophotometric analysis of archival tumor specimens used in retrospective studies were evaluated quantitatively. May-Grünwald-Giemsa-stained cytologic slides up to 20 years old could be restained by the Feulgen reaction with excellent results if they were destained in methanol and refixed in formaldehyde prior to Feulgen staining. Storage time had only a minor influence on Feulgen stainability. However, a considerable variation in the intensity of the Feulgen stain was observed between different slides stained simultaneously; this variation was not related to storage time. As a consequence of this variation, the use of internal staining controls, such as granulocytes, is an absolute necessity in the quantitative comparison of different slides. By expressing DNA data from the tumor cells in relative values (c values) related to the internal staining control (with a defined mean value of 2c), Feulgen ploidy level determinations could be made as accurately from measurements on old, destained slides as on cells obtained from fresh tumor material. The ploidy level could also be accurately determined in most cases of prostatic carcinoma from measurements on histopathologic sections.     

Assessment of quantitative techniques in paleobiogeography

A series of multivariate methods has been compared to assess their effectiveness in extracting essential information out of a complex micropaleontological data-set. The data-set used for this experiment consists of relative frequencies (percentages) of Miocene coccolith taxa or groups of taxa in cores of the Deep Sea Drilling Project (DSDP) from the Atlantic Ocean. All methods tested are varieties of principal components analysis in R- and Q-mode, and “true” factor analysis. Various secondary rotational procedures ancillary to some of these methods are also tested.A test, denoted Δ-Test, is developed, which assesses how well principal components or factors reproduce the data-set. Δ-Test may be used for determining the optimum number of principal components or factors, the most relevant rotational procedure, and thus the most suitable analytical technique. The Δ-Test does not rely on mathematical testing, but on simple inspection of the compositions of the principal components or factors, and their relations to correlations existing in the data-set.Our experiment reveals that the most efficient methods are the maximum-likelihood factor analysis and the R-mode principal components analysis, within which the varimax (orthogonal) rotations best reproduce correlations. Of these methods, maximum-likelihood factor analysis is considered the optimum method, because of the greater simplicity of compositions. In addition to these methods, Kaiser's second generation “Little Jiffy” factor analysis was also found to be efficient. Three methods provide less sensitive reduction of the data: the “true” R-mode principal components analysis (without secondary rotations), the Q-mode principal components analysis, and the correspondence analysis.