bluuebunny/crossref_metadata_2025_split huggingface dataset:
The coefficient of error of optical fractionator population size estimates: a computer simulation comparing three estimators
Authors: Glaser E. M. , Wilson P. D. 
Published: March 2003

The optical fractionator is a design‐based two‐stage systematic sampling method that is used to estimate the number of cells in a specified region of an organ when the population is too large to count exhaustively. The fractionator counts the cells found in optical disectors that have been systematically sampled in serial sections. Heretofore, evaluations of optical fractionator performance have been made by performing tests on actual tissue sections, but it is difficult to evaluate the coefficient of error (CE), i.e. the precision of a population size estimate, by using biological tissue samples because they do not permit a comparison of an estimated CE with the true CE. However, computer simulation does permit making such comparisons while avoiding the observational biases inherent in working with biological tissue.This study is the first instance in which computer simulation has been applied to population size estimation by the optical fractionator. We used computer simulation to evaluate the performance of three CE estimators. The estimated CEs were evaluated in tests of three types of non‐random cell population distribution and one random cell population distribution. The non‐random population distributions varied by differences in ‘intensity’, i.e. the expected cell counts per disector, according to both section and disector location within the section. Two distributions were sinusoidal and one was linearly increasing; in all three there was a six‐fold difference between the high and low intensities. The sinusoidal distributions produced either a peak or a depression of cell intensity at the centre of the simulated region. The linear cell intensity gradually increased from the beginning to the end of the region that contained the cells. The random population distribution had a constant intensity over the region. A ‘test condition’ was defined by its population distribution, the period between consecutive sampled sections and the spacing between consecutive sampled disectors. There were 1000 independently simulated cell populations for each test condition, and a ‘trial’ was conducted for each of these cell populations. In each trial we calculated the (unique) true CE of the population size estimate and the three CE estimates obtained by applying the Scheaffer–Mendenhall–Ott (SMO) and both Gundersen–Jensen (GJ) estimators. We compared the estimated CEs with the true CEs for each population distribution. We found that the CE estimates obtained by the SMO estimator were closer to the true CEs and had less scatter than those of the nugget‐modified GJ estimator. Both had small positive bias. The CE estimates obtained by the unmodified GJ estimator exhibited widely varying bias and large scatter. In all the population distributions we tested, the average true CE was very nearly proportional to 1/√Q¯T, where Q¯T is the average number of cells counted in the two‐stage systematic sample.
Photogrammetric calibration of a stereo light microscope
Authors: DANUSER G. 
Published: March 2003

Quantitative stereo light microscopy has been used in very rare cases to obtain geometric representations of microscopic objects. Although the instrument itself has limitations, most of them efficiently overcome by other 3D microscopes, it bears considerable advantages in observing and measuring dynamic scenes with multiple objects. The biggest asset of the stereo method is that the full 3D work space is imaged in one shot — a property which distinguishes stereo from all the scanning techniques in 3D microscopy. With the work presented in this paper, we contribute to making the stereo technique applicable to the numerous mensuration tasks in the microscopic domain, where its potential would be invaluable.We report the photogrammetric calibration of a common main objective lens type stereo light microscope. Such a calibration is the initial step in ensuring accurate measurements with this instrument. First, we derive a mathematical formulation of the imaging function and discuss the estimation of the parameters involved. Then, three main problems of the practical implementation of the framework are addressed: the finding of a calibration standard, the automatic measuring of the many image coordinates required, and the stabilization of the parameter estimation. Finally, results of various calibration runs are presented and analysed under different aspects. Among these, the most important is the accuracy of the calibrated instrument in measuring 3D positions and positional relationships. With a Zeiss Stemi 11, Achromat 1.6 × we achieve accuracies of 1‰ laterally and 1–2% axially relative to the volume of the work space. On the highest magnification level this corresponds to 700 nm and 1.8 μm, respectively.
Non‐coating fixation techniques or redundancy of conductive coating, low kV FE‐SEM operation and combined SEM/TEM of biological tissues
Authors: Jongebloed  , Stokroos  , Van Der Want  , Kalicharan  
Published: March 2003

Non‐coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea‐pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus‐producing goblet cells.These methods are used firstly to confirm that non‐coating prepared specimens can be embedded for TEM observation at 60–100 kV without loss of detail information, and these images can be compared with cryo‐FE‐SEM images of the same structure/tissue. Secondly, they show that specimens treated according non‐coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required. In this way a comparison of images of subsequent fresh fracture faces is possible without a decrease in information on detail, which otherwise could happen after subsequent coating layers required after standard fixation. Thirdly, they show that non‐coating methods can be used quite well with low accelerating voltages because the osmium–tannic acid complex in the specimen surface produces a large number of backscattered and secondary electrons in the surface layer, showing in particular surface phenomena. Fourthly, they show that with an optimal non‐coating preservation, in combination with a well‐balanced pre‐fixation mixture, preparation artefacts due to extraction and even dehydration and drying are minimized. This is compared with images of the organ of Corti hair cells treated with a so‐called three‐aldehyde pre‐fixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.
Three‐dimensional morphometry in scanning electron microscopy: a technique for accurate dimensional and angular measurements of microstructures using stereopaired digitized images and digital image analysis
Authors: Minnich  , Leeb  , Bernroider  , Lametschwandtner  
Published: March 2003

A method for accurate dimensional and angular measurements of microstructures analysed in the scanning electron microscope is described. The method considers central and parallel projections and involves (a) digital image acquisition of stereopaired images from the scanning electron microscope's photodisplay, (b) generation of 3D‐image representations, (c) setting of measuring points in the digitized stereopaired images, (d) computation of exact space coordinates (x/y/z) from the corresponding point coordinates (xL/yL; xR/yR), (e) determination of distances and angles between consecutive corresponding points using vector equations, and (f) transfer of computed data into spreadsheets of the data analysis software using dynamic data exchange with simultaneous graphical display of the frequency distribution of variables.Measurements performed on specimens with known dimensions (grid with 10 μm wide square meshes, polystyrene beads with 0.33 μm diameter) and angles (synthetic crystals of K(Al,Cr)[SO4], CuSO4.5H2O and NaCl) revealed a high accuracy in dimensional as well as angular measurements (total error 1 ± 0.5%).In Monte Carlo experiments the overall error was found to depend strongly on the size of the measured structure relative to the size of the measurement field (field width).
Quantitative analyses of the biomineralization of different hard tissues
Authors: Arnold S. , Plate U. , Wiesmann H. P. , Stratmann U. , Kohl H. , Höhling H. J. 
Published: March 2003

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy‐filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.
High resolution protein localization using soft X‐ray microscopy
Authors: Meyer‐Ilse W. , Hamamoto D. , Nair A. , Lelièvre S. A. , Denbeaux G. , Johnson L. , Pearson A. L. , Yager D. , Legros M. A. , Larabell C. A. 
Published: March 2003

Soft X‐ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X‐ray transmissive ‘water window’, where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X‐ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver‐enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X‐ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three‐dimensional information on the distribution of proteins in cells.
Multiphoton versus confocal high resolution z‐sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors
Authors: Drummond D. R. , Carter N. , Cross R. A. 
Published: March 2003

SummaryMultiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z‐axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.
Quantitative phase‐amplitude microscopy II: differential interference contrast imaging for biological TEM
Authors: McMahon P. J. , Barone‐Nugent E. D. , Allman B. E. , Nugent K. A. 
Published: March 2003

SummaryAlthough phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.
Measurement of nanosecond time‐resolved fluorescence with a directly gated interline CCD camera
Authors: Mitchell A. C. , Wall J. E. , Murray J. G. , Morgan C. G. 
Published: March 2003

SummaryCCD cameras coupled optically to gated image intensifiers have been used for fast time‐resolved measurements for some years. Image intensifiers have disadvantages, however, and for some applications it would be better if the image sensor could be gated directly at high speed. Control of the ‘charge drain’ function on an interline‐transfer CCD allows the sensor to be switched rapidly from an insensitive state. The temporal and spatial properties of the charge drain are explored in the present paper and it is shown that nanosecond time resolution with acceptable spatial uniformity can be achieved for a small commercial sensor. A fluorescence lifetime imaging system is demonstrated, based on a repetitively pulsed laser excitation source synchronized to the CCD control circuitry via a programmable delay unit.
Freeze substitution of high‐pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water
Authors: Walther P. , Ziegler A. 
Published: March 2003

SummaryBiological membranes are often poorly visible with the electron microscope after high‐pressure freezing and freeze‐substitution. The water content of the sample and of the substitution medium is one factor among others that strongly influences membrane visibility. In order to investigate this effect, high‐pressure frozen yeast cells, rat‐pancreas tissue and arthropod tissue were freeze‐substituted with and without adding water to the substitution medium. The visibility of the biological membranes was generally improved if the substitution medium contained 1–5% water. The effect was especially pronounced in yeast cells, where membrane visibility was poor after freeze‐substitution with water‐free medium but good after addition of 5% water to the substitution medium.
High‐resolution scanning electron microscopy of immunogold‐labelled cells by the use of thin plasma coating of osmium
Authors: Suzuki E. 
Published: March 2003

SummaryThe feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.
Quantitative polarized light microscopy
Authors: Massoumian F. , Juškaitis R. , Neil M. A. A. , Wilson T. 
Published: March 2003

SummaryWe describe a simple modification to a confocal microscope, which analyses the state of polarization of light emerging from the specimen so as to permit quantitative polarized light microscopy to be performed. The system uses a novel form of rotating analyser which, together with lock‐in detection, permits images to be obtained where the image contrast corresponds to both specimen retardance and orientation (e.g. in the case of a birefringent specimen). Images are presented from a wide range of specimens and the origin of the contrast observed from simple point scatterers is investigated both theoretically and experimentally.
PACAP Increases the Cytosolic Ca2−/Concentration and Stimulates Somatodendritic Vasopresson Release in Rat Supraoptic Neurons
Authors: Shibuya Izumi , Noguchi Jun , Tanaka Keiko , Harayama Nobuya , Inoue Yoshitaka , Kabashima Narutoshi , Ueta Yoichi , Hattori Yukio , Yamashita Hiroshi 
Published: March 2003

Pituitary adenylate cyclase activating polypeptide (PACAP)‐like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10−12to 10−7M increased [Ca2+]iin dissociated SON neurons in a dose‐dependent manner. The patterns of the PACAP‐induced [Ca2+]iincrease were either sustained increase or cytosolic Ca2+oscillations. PACAP (10−7M) increased [Ca2+]iin 27 of 27 neurons and glutamate (10−4M) increased [Ca2+]iin 19 of 19 SON neurons examined, whereas angiotensin II (10−7M) increased [Ca2+]iin only 15 of 60 SON neurons examined. PACAP at lower concentrations (10−10to 10−8M) increased [Ca2+]iin 70–80% of neurons examined. Although the onset and recovery of the PACAP‐induced [Ca2+]iincrease were slower than those observed with glutamate, the spatial distribution of the [Ca2+]iincreases in response to the two ligands were similar: [Ca2+]iincrease at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP‐induced [Ca2+]iresponseswere abolished by extracellular Ca2+removal, thel‐type Ca2+‐channel blocker, nicardipine, or by replacement of extracellular Na+with N‐methyld‐glucamine, and were partially inhibited by the Na+‐channel blocker, tetrodotoxin. The N‐type Ca2+‐channel blocker,ω‐conotoxin GVIA did not significantly inhibit the PACAP‐induced [Ca2+]iresponses. Furthermore, PACAP (10−7M) as well as glutamate (10−4M) increased AVP release from SON slice preparations, and extracellular Ca2+removal or nicardipine inhibited the AVP release in response to PACAP.These results indicate that PACAP enhances Ca2+entry via voltage‐gated Ca2+channels and increases [Ca2+]i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.
Pharmacological Evidence that Prolactin Acts from Late Gestation to Promote Maternal Behaviour in Rabbits
Authors: González‐Mariscal G. , Melo A. I. , Parlow A. F. , Beyer C. , Rosenblatt J. S. 
Published: March 2003

AbstractWe investigated the role of prolactin and suckling stimulation in the expression of maternal behaviour of primiparous rabbits. Bromocriptine (1 mg/kg/day), given to intact mothers across postpartum days 1–5, decreased serum concentrations of prolactin to undetectable levels, reduced crouching, and increased time inside the nest. Failure of maternal nest‐building, provoked by bromocriptine injections from pregnancy day 26 to parturition or to postpartum day 5, correlated with a stronger reduction in crouching and an increased time inside the nest, measures of disturbed maternal behaviour, on postpartum days 3 and 5. Preventing suckling by thelectomy did not prevent prolactin release but reduced crouching incidence and increased the time spent inside the nest on postpartum days 3 and 5. Bromocriptine, injected in thelectomized mothers across postpartum days 1–5, further reduced the incidence of crouching and increased the time spent inside the nest on postpartum days 3 and 5. We conclude that prolactin acting prepartum facilitates maternal behaviour initiation in rabbits and, together with pup stimulation, maintains this behaviour across lactation.
The In Vitro Role of Tumour Necrosis Factor‐Alpha and Interleukin‐6 in the Hypothalamic‐Pituitary Gonadal Axis
Authors: Russell S. H. , Small C. J. , Stanley S. A. , Franks S. , Ghatei M. A. , Bloom S. R. 
Published: March 2003

AbstractThe adipocyte derived hormone leptin has been implicated as an important nutritional signal to the reproductive system, but the role of other adipocyte related cytokines is not clear. Tumour necrosis factor‐alpha (TNF‐α) and interleukin (IL)‐6 are present in adipose tissue and released into the circulation where plasma levels correlate positively with body mass index and body fat mass. These cytokines could play a role in signalling nutritional status to the hypothalamic‐pituitary‐gonadal axis. We investigated the effects of TNF‐α and IL‐6 on basal and luteinizing hormone releasing hormone (LHRH) stimulated luteineizing hormone (LH) release from cultured anterior pituitary cells, harvested from either proestrus female or male Wistar rats. We examined the effects of TNF‐α and IL‐6 on LHRH release from hypothalamic explants harvested from proestrus female and male rats in vitro. IL‐6 significantly suppressed LHRH stimulated LH release from male dispersed pituitaries throughout the dose range, but did not influence basal LH release. IL‐6 had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from proestrus females. By contrast, TNF‐α significantly suppressed LHRH stimulated LH release in dispersed pituitaries from proestrus female rats in a dose responsive manner, but did not influence basal LH release. TNF‐α had no effect on basal or LHRH stimulated LH release in dispersed pituitaries from male rats. TNF‐α and IL‐6 had no effect on LHRH release from male hypothalamic explants in vitro. TNF‐α and IL‐6 had no effect on LHRH release from proestrus female hypothalamic explants in vitro. TNF‐α and IL‐6 have differential effects in dispersed pituitaries harvested from males and proestrus female rats. TNF‐α and IL‐6 may be important in mediating some of the nutritional effects on the reproductive axis by acting at the level of the anterior pituitary rather than the hypothalamus.