bluuebunny/crossref_metadata_2025_split huggingface dataset:
Estimating the length of a bounded curve in three dimensions using total vertical projections
Authors: Cruz‐Orive Luis M. , Howard* C. Vyvyan 
Published: August 2011

SUMMARYA new method is proposed to estimate the total length of a bounded, isolated linear feature in three dimensions from ‘total vertical projections’, obtained by rotating the curve about a fixed axis (arbitrarily called ‘vertical’) and projecting it onto a fixed vertical plane. No sections are required. Properly stained and embedded neuron dendrites, mycelial trees, fluorescent cytoskeletal filaments within a cell, etc., are candidate specimens for the method, especially in combination with the new devices for non‐invasive three‐dimensional microscopy. It is necessary that the specimen curve is rigid (i.e. of constant shape), that its length density is not too high (so that overlapping effects are not important) and that the embedding medium is fairly transparent. Given these requirements, the method can be very accurate and convenient to use, as is exemplified here.
An evanescent‐field optical microscope
Authors: van Hulst N. F. , de Boer N. P. , Bölger B. 
Published: August 2011

SUMMARYThe classic diffraction limit of resolution in optical microscopy (∼γ/2) can be overcome by detecting the diffracted field of a submicrometre‐size probe in its near field. The present stage of this so‐called scanning near‐field optical microscopy (SNOM) is reviewed.An evanescent‐field optical microscope (EFOM) is presented in which the near‐field regime is provided by the exponentially decaying evanescent field caused by total internal reflection at a refractive‐index transition. A sample placed in this field causes a spatial variation of the evanescent field which is characteristic for the dielectric and topographic properties of the sample. The evanescent field is frustrated by a dielectric probe and thus converted into a radiative field.In our case the probe consists either of an etched optical fibre or of a highly sharpened diamond tip. The probe is scanned over the sample surface with nanometre precision using a piezo‐electric positioner. The distance between probe and sample is controlled by a feedback on the detected optical signal. The resolution of the microscope is determined by both the gradient of the evanescent field and the sharpness of the tip. Details of the experimental set‐up are discussed.The coupling of the evanescent field to the submicrometre probe as a function of probe‐sample distance, angle of incidence and polarization has been characterized quantitatively. The observed coupling is generally in agreement with presented theoretical calculations.Microscopy has been performed on a regular latex sphere structure, which clearly demonstrates the capacity of the evanescent‐field optical microscope for nanometre‐scale optical imaging. Resolution is typically 100 nm laterally and 10 nm vertically.The technique is promising for biological applications, especially if combined with optical spectroscopy.
The point‐spread function of a confocal microscope: its measurement and use in deconvolution of 3‐D data
Authors: Shaw Peter J. , Rawlins David J. 
Published: August 2011

SUMMARYWe have measured the point‐spread function (PSF) for an MRC‐500 confocal scanning laser microscope using subresolution fluorescent beads. PSFs were measured for two lenses of high numerical aperture—the Zeiss plan‐neofluar 63 × water immersion and Leitz plan‐apo 63 × oil immersion—at three different sizes of the confocal detector aperture. The measured PSFs are fairly symmetrical, both radially and axially. In particular there is considerably less axial asymmetry than has been demonstrated in measurements of conventional (non‐confocal) PSFs. Measurements of the peak width at half‐maximum peak height for the minimum detector aperture gave approximately 0·23 and 0·8 μm for the radial and axial resolution respectively (4·6 and 15·9 in dimensionless optical units). This increased to 0·38 and 1·5 μm (7·5 and 29·8 in dimensionless units) for the largest detector aperture examined. The resulting optical transfer functions (OTFs) were used in an iterative, constrained deconvolution procedure to process three‐dimensional confocal data sets from a biological specimen—pea root cells labelled in situ with a fluorescent probe to ribosomal genes. The deconvolution significantly improved the clarity and contrast of the data. Furthermore, the loss in resolution produced by increasing the size of the detector aperture could be restored by the deconvolution procedure. Therefore for many biological specimens which are only weakly fluorescent it may be preferable to open the detector aperture to increase the strength of the detected signal, and thus the signal‐to‐noise ratio, and then to restore the resolution by deconvolution.
Digital three‐dimensional visualization of cellular organelles studied by medium‐ and high‐voltage electron microscopy
Authors: Beorchia A. , Ploton D. , Menager M. , Lebonvallet S. , Thiry M. , Bonnet N. 
Published: August 2011

SUMMARYTilted thick sections (one‐half to several micrometres) of biological specimens observed with medium‐ to high‐voltage electron microscopes are extremely useful for the study of the three‐dimensional (3‐D) structure of organelles.If high resolution in 3‐D visualization and 3‐D reconstruction is needed, many images corresponding to various angles of rotation and tilt must be recorded. This necessitates very time‐consuming work—including eventual photographic processing—before good positioning of the object is defined.We have developed software which permits very rapid and precise determination of the tilt‐axis, the registration of tilted views, 3‐D measurements and 3‐D visualization. Images are digitized either from negative films or directly with a camera fitted to the microscope. The application of the software is performed in minutes and allows for a rapid check of the quality of the tilt‐series and of the features of interest of the object.Application of the software to the study of the 3‐D structure of active components of the nucleolus stained with silver is shown.
SEM electron channelling patterns as a technique for the characterization of ion‐implantation damage
Authors: Page Trevor F. , McHargue Carl J. , White C. W. 
Published: August 2011

SUMMARYElectron channelling patterns (ECPs) formed in back‐scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth).Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back‐scattering (RBS). Selected‐area ECPs were obtained in a Jeol‐840 electron microscope operating over the range 5–40 kV in 5‐kV steps.Progressive ECP degradation—in terms of high‐order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near‐surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (∼ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near‐surface structural damage.
Determination of the Cnl parameter in the Bethe formula for the ionization cross‐section by the use of Cliff‐Lorimer KAB factors obtained at different accelerating voltages in a TEM
Authors: Gauvin R. , L'Espérance G. 
Published: August 2011

SUMMARYThere is a clear advantage in using theoretical KAB factors to perform quantitative microanalysis, because they do not have to be experimentally determined. To compute KAB factors, we need a very good parameterization of ionization cross‐sections. If the bnl parameter in the Bethe formula for the ionization cross‐section is not a function of the element (A or B), then only the Cnl parameter is required to compute KAB factors. In this paper we present a new method to measure Cnl based on the ratio of KAB factors obtained at different accelerating voltages.
A modified Jelley refractometer
Authors: Langford Stephen A. 
Published: August 2011

SUMMARYLight beams are read directly (not as virtual images) against a scale erected about 74 cm in front of the prism, with a modified Jelley refractometer. Liquid temperatures are controlled by thermistors placed at each end of the prism trough, which is enclosed in an aluminium block.The scale is calibrated against the refractive index (RI) of distilled water, seven (sic) RI estimates for six Cargille liquids related to six glass standards, and the label RIs of ninety‐eight other Cargille liquids.The calibrated refractometer is used accurately and precisely to calibrate ninety‐six Cargille liquids, over the inclusive 1ċ400–1ċ800 (25ċ0°C, 589ċ3 nm, RI unit) label range, at liquid temperatures of 19–49°C and illuminating wavelengths of 435ċ8–694ċ3 nm.
Axial resolution of confocal microscopes with parallel‐beam detection
Authors: Shao Zhifeng , Baumann Otto , Somlyo Andrew P. 
Published: August 2011

SUMMARYWe present a simple theory for the evaluation of the axial resolution of a confocal scanning microscope with parallel‐beam detection. The results demonstrate that, in certain cases, the collection efficiency is low compared with a conventional confocal microscope, but the axial resolution may be further improved.
Electron energy‐loss spectroscopy analysis of adriamycin‐plasma membrane interaction
Authors: Diociaiuti M. , Molinari A. , Calcabrini A. , Arancia G. 
Published: August 2011

SUMMARYOur previous studies on the mechanism of cytotoxic action of the anti‐tumour drug adriamycin (ADR) indicated that this anthracyclinic antibiotic strongly modified the molecular architecture of the plasma membrane of human erythrocytes, presumably becoming incorporated within both lipid layers. In order to verify this hypothesis, electron energy‐loss spectroscopy (EELS) has been used to compare the P content in control and ADR‐treated erythrocyte ghosts. EELS measurements allowed us to reveal a significant reduction in the P/C ratio in erythrocyte ghosts after ADR treatment. This finding seems to reflect a phospholipid ‘dilution’ produced by the incorporation of the drug molecules in the membrane layers. A structural model of the ADR‐membrane interaction is proposed.
Quantitative theory of ideal phase‐contrast microscopy, taking object width into account
Authors: Goldstein D. J. 
Published: August 2011

SUMMARYIn ‘ideal’ phase‐contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase‐contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field.The absolute brightness of an image in ideal central dark‐field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field.The equations give results for ideal phase‐contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non‐ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination.
The experimental effect of detector size on confocal lateral resolution
Authors: Glass Monty , Dabbs Tim 
Published: August 2011

SUMMARYThe lateral resolution of a scanning‐spot microscope has been measured as the product of the pin‐hole size and lens numerical aperture of the detector approaches the confocal limit. The resolution follows an earlier prediction, improving approximately 30% in going from that of a conventional scanning‐spot or broad‐viewing‐area microscope to that of a fully confocal microscope. The discrepancy between the data and the theoretical curve can be attributed to lens apodization arising from the use of multi‐element thick lenses and a non‐ideal, truncated Gaussian beam profile.
Microscopy of cell cultures through thin plastic films
Authors: Moroz Pavel E. 
Published: August 2011

SUMMARYMicroscopy of cells growing in vessels containing plastic films as a substrate or as a transparent window is facilitated by a contact cap on the objective or the contact objectives for intravital microscopy. When applied to microscopical examination of living cells through a thin film, the cap considerably improves the conditions of observation with high‐power dry objectives and makes it possible to use water‐ and oil‐immersion objectives.
A device to ease grid coating for EM autoradiography
Authors: Riboni Laura , Núñez‐Durán Harol 
Published: August 2011

SUMMARYA modification to the standard device used to hold grids during the process of coating with emulsion for EM autoradiography is described. The device has a plastic base with six bronze poles attached. Each pole has a groove machined along its side to the top, and a shallow depression at the end of the groove. Instructions for the construction of the poles and for use of the device are given. The innovative design allows the user to feel the top of the pole, and thus simplifies selection of the coated grids. As grids are less exposed to safe light than in the standard procedure, the background of silver grains decreases; also the chance of missing a grid decreases.
Freeze‐substitution techniques for preparing nematodes for scanning electron microscopy
Authors: Wharton D. A. 
Published: August 2011

SUMMARYThe effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO4/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.
Image registration and distortion correction in ion microscopy
Authors: Olivo J. C. , Kahn E. , Halpern S. , Fragu P. 
Published: August 2011

SUMMARYWe present a method whereby the geometric registration of a series of ion microscopic images is performed by applying a two‐step procedure. After applying a global linear transformation that corrects for geometric differences, a non‐linear elastic transform is used in order to match local properties and structures of the images. Transformation parameters are computed on the basis of shape‐specific points in the images. Distortion correction is achieved by relating ion images to the optical image of the same field and by using the two‐step algorithm to register the images. This methodology is evaluated on synthetic misaligned objects and on thyroid tissue images.
Real‐time quantitative elemental analysis and mapping: microchemical imaging in cell physiology
Authors: LeFurgey A. , Davilla S. D. , Kopf D. A. , Sommer J. R. , Ingram P. 
Published: August 2011

SUMMARYRecent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X‐ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics‐based microcomputer to acquire spectrally filtered X‐ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post‐acquisition image map with regard to specimen drift. Both high‐resolution quantitative energy‐dispersive X‐ray images of freeze‐dried cyrosections and low‐dose quantitative bright‐field images of frozen‐hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on‐line in real time. In addition, the unified design of the software program provides for off‐line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer‐aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure‐function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell‐to‐cell interactions.
Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle
Authors: Dalen H. , Scheie P. , Nassar R. , High T. , Scherer B. , Taylor I. , Wallace N. R. , Sommer J. R. 
Published: August 2011

SUMMARYSingle, intact, frog skeletal muscle fibres and whole frog hearts were quick‐frozen on a polished, liquid‐He‐cooled copper block and examined in the electron microscope after freeze‐substitution and freeze‐fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze‐substituted preparations were confirmed by freeze‐fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoarte‐facts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non‐muscle cells as well.
An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation
Authors: Hanyu Yoshiro , Ichikawa Michinori , Matsumoto Gen 
Published: August 2011

SUMMARYThe metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light‐mass plunger and a recently developed β‐gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.
The effects of glycol methacrylate as a dehydrating agent on the dimensional changes of liver tissue
Authors: Gerrits Peter O. , Horobin Richard W. , Stokroos Ietse 
Published: August 2011

SUMMARYThe dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration.The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.
Non‐raster isotropic scanning for analytical instruments
Authors: Sasov A. 
Published: August 2011

SUMMARYA new type of image‐forming, non‐raster isotropic scanning is presented. Isotropic scanning may be used in electron microscopy, for TV systems, image processing equipment, etc. With this approach it is possible to obtain less distorted images than with raster scanning, and more reliable quantitative characteristics in the image processing systems.