bluuebunny/crossref_metadata_2025_split huggingface dataset:
A particle segmentation method based on nucleation and growth of the background
Authors: ZHENG XIN , KOLB MAX 
Published: August 2011

SUMMARYA new algorithm for image segmentation is proposed, which is capable of extracting particles in the presence of noise and background fluctuations. It begins with the detection of small regions belonging to the background, called background nuclei, and then lets these nuclei grow to become the entire background. Edge information and region information of the image are used simultaneously.
Atomic force microscopy of freeze‐fracture replicas of rat atrial tissue
Authors: KORDYLEWSKI L. , SANER D. , LAL R. 
Published: August 2011

SummaryAtomic force microscopy (AFM) has provided three‐dimensional (3‐D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well‐established technique of transmission electron microscopy (TEM) was performed. Freeze‐fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large‐scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3‐D surface contour) and TEM (2‐D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.
Calibration of the scanning (atomic) force microscope with gold particles
Authors: XU S. , ARNSDORF M. F. 
Published: August 2011

SummaryScanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.
Contrast in the transmission mode of a low‐voltage scanning electron microscope
Authors: GOLLA U. , SCHINDLER B. , REIMER L. 
Published: August 2011

SummaryThe contrast thicknesses (xk) of thin carbon and platinum films have been measured in the transmission mode of a low‐voltage scanning electron microscope for apertures of 40 and 100 mrad and electron energies (E) between 1 and 30 keV. The measured values overlap with those previously measured for E (≥ 17keV) in a transmission electron microscope. Differences in the decrease of xk with decreasing E between carbon and platinum agree with Wentzel‐Kramer‐Brillouin calculations of the elastic cross‐sections. Knowing the value of xk allows the exponential decrease ∝ exp(—x/xk) in transmission with increasing mass‐thickness (x = ρt) of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and darkfield modes.
Volume and surface area measurement of viable chondrocytes in situ using geometric modelling of serial confocal sections
Authors: GUILAK F. 
Published: August 2011

SummaryThis study describes a technique for noninvasive determination of the surface area and volume of chondrocytes using the confocal scanning laser microscope, and the fundamental limitations associated with its application. Using geometric modelling principles, an isointensity surface contour was formed from a series of optical sections recorded with the confocal microscope. Using a combined surface‐ and volume‐based algorithm, the surface area, volume and other morphometric descriptions were calculated from a polygonal description of the cell surface. The high image contrast required for repeatable identification of the cell border was achieved through the use of a fluorescent dye, which was excluded from cells by an intact membrane. Calibration results indicated that the theoretical modelling algorithm is relatively precise when applied to simulated convex (ellipsoidal) cells, with overall errors of less than 0·5% in surface area and volume measurements. When applied to low‐noise, high‐contrast volume data recorded on the confocal microscope, typical coefficients of variation of 2–4% were determined for length measurements, 2–5% for volume measurements and 3–6% for surface area measurements either for latex microspheres or for chondrocytes. While the precision of the method is comparable to standard histological techniques, its accuracy is difficult to assess, as systematic errors are unpredictable and may be introduced from several sources.
Accurate alignment of sets of images
Authors: SAXTON W. O. 
Published: August 2011

SummaryTwo modifications to the conventional procedure of cross‐correlation are described, widely used for establishing the relative alignment of the members of a set of images from which a higher resolution or more interpretable restoration is sought. Both achieve a high and sharp peak in circumstances where the conventional peak is too ill defined to be recognizable; neither involves significant additional computation time. The more general method requires rough knowledge of the imaging conditions, but a variant applicable to images with axial resolution has no such requirement. In addition, a least‐squares procedure is presented for achieving an optimum compromise between many pair‐wise displacement measurements, preventing the accumulation of alignment errors across a set of images.
Cryo automated electron tomography: Towards high‐resolution reconstructions of plastic‐embedded structures
Authors: BRAUNFELD M. B. , KOSTER A. J. , SEDAT J. W. , AGARD D. A. 
Published: August 2011

SummaryThe use of fully automated data collection methods for electron tomography allows a substantial reduction in beam dose. The goal has been to develop new protocols for data collection defining optimal approaches for maintaining data self‐consistency and maximizing the useful resolution of the reconstruction. The effects of irradiation and post‐cure microwaving were examined for a variety of embedding media (Epon, Epox, Lowicryl) in order to quantify beam damage with the goal of identifying the most beam stable embedding medium. Surprisingly, the substantial dose reduction made possible by automated data collection did not result in a significant decrease in specimen shrinkage even for samples stabilized by preirradiation. We believe that the accelerated shrinkage is a direct consequence of the stroboscopic illumination patterns inherent to automated data collection. Further‐more neither the choice of embedding resin nor microwave post‐curing greatly affected shrinkage. Finally, cryogenic data collection was investigated as a means to minimize the effects of secondary radiation damage. Minimal preirradiation coupled with low‐temperature automated data collection greatly reduces shrinkage and should result in high‐quality data for three‐dimensional reconstructions.
A novel method for mean cell volume estimation
Authors: WEBSTER P. , GRIFFITHS G. 
Published: August 2011

SummaryA novel method is described for the estimation of the mean cell volume of cell populations grown in suspension. The cells are filtered onto a nitrocellulose filter to form a cylindrical pellet which is embedded in epoxy resin. Using estimates of pellet height and radius, the number of cells in the pellet and of the volume density of the cells in the pellet, it is possible to produce an unbiased estimate of the mean cell volume. This method is compared, using cell suspensions of the blood parasite Trypanosoma brucei, with mean cell volume estimation using a Coulter channellizer. A Coulter channellizer was also used to compare the mean cell volume of living trypanosomes with that of aldehyde‐fixed populations, and the values obtained were compared with those obtained using the new method. The estimated mean cell volume of a T. brucei clone was used to derive values from volume densities obtained by point and intersection counts for the absolute volumes of the flagellar pocket, the nucleus, and endocytic organelles containing internalized horseradish peroxidase and transferrin‐gold after 30‐min incubations at 310 K. Estimated values for the surface area of the flagellar pocket and the surface area of a cell were also obtained. From known data on the total amount of variant surface glycoprotein molecules per cell and the known packing density of membrane proteins, it was estimated that approximately 80% of the molecules must reside in intracellular compartments. It was estimated that the equivalent of 5% of the surface membrane may be internalized per minute, an amount which is almost the size of the entire flagellar pocket membrane.
Astigmatism of a thick cylindrical object in reflective mode CSLM
Authors: WANG CHANG‐GUI , RÄIKKÖNEN H. , LUUKKALA M. 
Published: August 2011

SummaryThe axial response of a basic confocal microscope is determined when the sample is a thick cylindrical or tubular structure. The response from the back wall of the cylindrical sample is split into two separate signals due to basic aberration or astigmatism effects.
ESI in situ analyses of extrachromosomal RNP granules
Authors: ABOLHASSANI‐DADRAS S. , VÁZQUEZ‐NIN G. H. , ECHEVERRÍA O. M. , ROUELLE‐ROSSIER V. BOUTINARD , FAKAN S. 
Published: August 2011

SummaryObservation of unstained ultrathin sections of salivary gland cells of Chironomus thummi and C. tentans, by means of electron spectroscopic imaging (ESI), has revealed that phosphorus is distributed in two types of granular structures in the nucleoplasm of these cells. In addition to a specific type of premessenger ribonucleoprotein (RNP) particle, known as the Balbiani ring (Br) granule, ESI revealed a new type of phosphorus‐rich small granular component. Examination of unstained sections by energy‐filtering transmission electron microscopy (EFTEM) offers the opportunity of obtaining the signal from the specimen alone, thus avoiding the possible contributions of heavy metals present in any staining product.
A high‐sensitivity CCD system for parallel electron energy‐loss spectroscopy (CCD for EELS)
Authors: TANG Z. , HO R. , XU Z. , SHAO Z. , SOMLYO A. P. 
Published: August 2011

SummaryA cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy‐loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal‐to‐noise ratio and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorus L edge generated from a phosphoprotein, Phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required for elemental mapping at a given sensitivity.
Second‐order stereology for pores in translucent alumina studied by confocal scanning laser microscopy
Authors: KARLSSON L. M. , LILJEBORG A. 
Published: August 2011

SummaryThe three‐dimensional (3‐D) arrangement of pores in translucent alumina was investigated with a confocal scanning laser microscope (CSLM). By moving the focal plane of the CSLM down into the material, a stack of serial thin optical sections was obtained to produce a 3‐D image of the pores. Computer‐based image analysis was used to obtain the coordinates of the pore centroids. The distance distribution function G(r) and the second‐order functions K(r), L(r), H(r) and g(r) were used to analyse the spatial point pattern of the pore centroids. Estimates of the preceding functions obtained from eight stacks of sections were compared with the corresponding functions for a 3‐D stationary Poisson point process, which served as a reference model for complete spatial randomness. The analysis suggested that the pore centroids were arranged in an aggregated pattern within a range of about 10 μm.
Modelling of vascular growth processes: A stochastic biophysical approach to embryonic angiogenesis
Authors: SANDAU K. , KURZ H. 
Published: August 2011

SummaryIn a computer simulation, growth of a capillary network is driven by a stochastic process on a planar hexagonal grid. Starting at a point source, the probabilities for the formation of new capillary elements depend on local biophysical knowledge. This knowledge is mainly derived from the flow theorem of Hagen–Poiseuille and the diameter exponent Δ. The hexagonal grid is visualized as being supported by a cylinder or a sphere. An arterial tree results from the adaptive diameter augmentation, and is considered to have limited fractal properties. The dimension of its border, and the time course of growth and of blood pressure are compared with biological data from the chorioallantoic membrane (CAM) of incubated chicken eggs. The model is discussed in view of mechanosensitivity and cell–matrix interactions of endothelial cells, and CAM haemodynamics.
Retention of vacuole contents of plant cells during fixation
Authors: DONG Z. , McCULLY M. E. , CANNY M. J. 
Published: August 2011

SummaryChanges in the semi‐permeability of tonoplast during fixation were studied using beetroot tissue. Using vacuole betacyanin as the indicator, the permeability of the tonoplast was assessed by the leakage of this pigment as determined by changes in the optical density of the solution bathing the tissue. Cryo‐analytical scanning electron microscopy was used to monitor the changes in ion concentration in the cells during fixation. Fixatives were 3% glutaraldehyde or 4% formaldehyde in 0·025 m phosphate buffer at room temperature or on ice. Results showed that glutaraldehyde, especially at low temperature, takes as long as 30 h to disrupt the semi‐permeability of the tonoplast of beetroot cells, while in formaldehyde on ice, these cells begin to lose their selective permeability in 15 min. This study confirms that the semi‐permeability of the tonoplast may not be lost until long after the cytoplasm has been fixed and suggests that this explains why cold fixation in 3% glutaraldehyde for about 12 h has become the most reliable standard procedure for the successful preservation of vacuolated plant cells.
Central and peripheral nervous structures as seen at the confocal scanning laser microscope
Authors: CASTANO P. , MARCUCCI A. , JR A. MIANI , MORINI M. , VERALDI S. , RUMIO C. 
Published: August 2011

SummaryCentral neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi‐Cox mercuric impregnation, however, provides specimens which are precipitate‐free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He–Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.
Rapid estimation of bacterial antibiotic susceptibility with flow cytometry
Authors: MASON D. J. , ALLMAN R. , STARK J. M. , LLOYD D. 
Published: August 2011

SummaryBacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol and observing changes in low‐angle light scatter (excitation light scattered by up to 15°). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2–5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady‐state growth in liquid culture.
Rapid assay for pathogenic salmonella organisms by immunofluorescence flow cytometry
Authors: PINDER A. C. , MCCLELLAND R. G. 
Published: August 2011

SummaryMulti‐parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full‐fat milk). In all cases, the method was accurate to levels of below 104 target cells per ml for a total assay time of about 30 min. After 6 h non‐selective enrichment in the presence of a 10 000‐fold excess of competing micro‐organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml−1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro‐organisms in the food industry.
Fractal characterization by frequency analysis: III. Effect of noise
Authors: PANCORBO M. , ANGUIANO E. , AGUILAR M. 
Published: August 2011

SummaryThe effect of noise in the fractal characterization by frequency analysis of surface images obtained by scanning tunnelling microscopy (STM), atomic force microscopy (AFM) or profilometry has been studied. The origin of noise and its relationship to the signal is discussed. A procedure to simulate noisy images is presented. From the study it is concluded that the method usually used to characterize noise in STM is not valid and it is shown that fractal characterization of surfaces when noise is present by traditional frequency analysis methods is not possible. A new method to perform both the noise characterization and the fractal characterization of surfaces when noise is present is proposed.
Vitrification of aqueous suspensions from a controlled environment for electron microscopy: An improved plunge‐cooling device
Authors: BATTERSBY B. J. , SHARP J. C. W. , WEBB R. I. , BARNES G. T. 
Published: August 2011

SummaryIn the process of vitrifying aqueous suspensions for cryotransmission electron microscopy, water is solidified without crystallization. Vitrification can be achieved by rapidly plunging an aqueous thin film into a liquid cryogen. The preparation of aqueous thin films prior to vitrification must be performed in an environmental cabinet at controlled temperature and humidity in order to prevent evaporation and temperature‐induced phase changes in the thin film. The device described here incorporates several important features which make the apparatus simpler and more convenient to use than similar devices described in the literature. One of these features includes the use of a totally enclosed environmental cabinet in which the grid, sample, micropipette and absorbent paper are equilibrated before thin‐film preparation. Other features include a cryogen dewar on a swing arm for easy refilling, a guillotine shutter which is used to trigger the plunger electrically and a semiautomatic system which facilitates rapid transfer of the vitrified specimen from liquid propane to liquid nitrogen for storage and reduces handling of the specimen. To demonstrate the utility of the device, results showing the influence of temperature on the morphology of phospholipid vesicles are presented. A commercial cryotransfer apparatus (which is used for transportation of the vitrified specimen to the electron microscope cold‐stage) has been modified to reduce the possibility of reversion of the vitreous phase to the crystalline ice phases.