bluuebunny/crossref_metadata_2025_split huggingface dataset:
Image sharpness and contrast transfer in coherent confocal microscopy
Authors: OLDENBOURG R. , TERADA H. , TIBERIO R. , INOUÉ S. 
Published: August 2011

SummaryConfocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non‐confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non‐confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non‐confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non‐confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non‐confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non‐confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.
A light‐emitting diode light standard for photo‐ and videomicroscopy
Authors: BEACH J. M. , DULING B. R. 
Published: August 2011

SummaryA light calibration system consisting of a compact light‐emitting diode (LED) source with feedback control of intensity is described. The source is positioned in the focal plane of the microscope objective and produces flat‐field illumination of up to 31 μW. The source can be easily used to determine the performance of microscope optics and camera response. It can also be used as a standard light source for calibration of experimental systems. Selectable light intensities are produced by controlling the LED input power via a feedback circuit consisting of a photodiode that detects output light intensity. Spectral coverage extends between 550 and 670nm using green, yellow and red LEDs mounted side by side, which are selected individually. The LED chips are encapsulated in plastic diffusers which homogenize the light, and a flat field of illumination is obtained through a thin 1‐mm‐diameter aperture positioned directly over each chip. Provision is made for insertion of Ronchi rulings over the aperture to enable measurements of contrast modulation in a uniform field. The light may be pulse‐modulated to assess camera response times and the device can be synchronized with video frames. Narrow bandpass interference filters can be placed between the objective lens and the LED source to produce monochromatic light without affecting the spacing of controlled light intensities since emission spectra do not shift appreciably over the range of LED powers chosen in this design. Results of tests using controlled light intensity and uniform illumination are presented.
Scanning luminescence X‐ray microscopy: Imaging fluorescence dyes at suboptical resolution
Authors: JACOBSEN C. , LINDAAS S. , WILLIAMS S. , ZHANG X. 
Published: August 2011

SummaryScanning luminescence X‐ray microscopy is based on the use of the very small focused probe of a scanning X‐ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X‐ray source and a Fresnel zone plate to produce a focused X‐ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X‐ray probe (the microscope has imaged features at 36‐nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye‐tagged sites of biochemical activity at the resolution of the X‐ray microscope in wet, unsectioned, and unfixed cells, especially with soft X‐ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye‐tagged features should be 2–22 times lower than it is in transmission X‐ray microscopy. A possible extension of the technique for three‐dimensional imaging at the transverse resolution of the X‐ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.
A device for picking up ultrathin serial sections
Authors: MEYER E. P. , DOMANICO V. J. 
Published: August 2011

SummaryReconstruction from serial sections is often required to understand the architecture of biological tissue. The sampling of serial sections calls for an enormous amount of patience and skill. Sections are cut with a diamond knife, sampled in a trough and must then be collected on grids for examination with the electron microscope (EM). This last step, in which the section ribbons have to be aligned with the EM grid, is especially difficult as both hands have to work in perfect co‐ordination. A simple manually operated or motorized mechanical device has been designed which facilitates the collection of EM section ribbons.
A compact Schwarzschild soft X‐ray microscope with a laser‐produced plasma source
Authors: HORIKAWA Y. , NAGAI K. , MOCHIMARU S. , IKETAKI Y. 
Published: August 2011

SummaryA compact Schwarzschild soft X‐ray microscope using a laser‐produced plasma soft X‐ray source has been developed. The laser‐produced plasma source, which is small but of high brilliance, has made it possible to use the soft X‐ray microscope in a small laboratory. The microscope is composed of a Schwarzschild objective and a grazing incidence mirror condenser. Image contrast for biological specimens in soft X‐ray regions is investigated briefly. It is possible to observe the fine structures of a thin specimen at a wavelength of 15 nm; at this wavelength high‐contrast images of biological specimens have been obtained with a single laser shot of pulse width of 8 ns at a resolution of 0·3 μm. The resolution of the system is limited by the detector.
An investigation of substrate and sample preparation effects on scanning tunnelling microscopy studies on xanthan gum
Authors: WILKINS M. J. , DAVIES M. C. , JACKSON D. E. , ROBERTS C. J. , TENDLER S. J. B. 
Published: August 2011

SummaryScanning tunnelling microscopy is developing as an important biophysical tool for the molecular imaging of biological material. In this study, the effect of sample deposition technique and substrate employed on the resultant images of a microbial polysaccharide is investigated. Scanning tunnelling microscopy topographs of xanthan gum, pipette deposited and spray deposited onto highly orientated pyrolytic graphite and mica substrates are presented. The use of pipette deposition of the aqueous sample solution is shown to result in a xanthan network. In contrast, images of isolated xanthan molecules are obtained when spray deposition with glycerol is employed. The effect of these deposition techniques on the macroscopic distribution of sample material across substrates is shown using confocal fluorescence microscopy.
Effects of noise and anisotropy on the determination of fractal dimensions
Authors: RUSS J. C. 
Published: August 2011

SummaryMeasurement of the fractal dimension of surfaces imaged by the scanning tunnelling microscope, atomic force microscope or similar instruments can be performed using several different algorithms. Dimensional analysis—plots of log (perimeter) vs. log (area)—are compared to Korcak and slit‐island methods, which also employ a horizontal plane section, and to Fourier analysis of the two‐dimensional array of elevation values. The effects of surface anisotropy and instrument noise on each of these measurement techniques is investigated.
Directional analysis of planar fibre networks: Application to cardboard microstructure
Authors: MOLCHANOV I. , STOYAN D. , FYODOROV K. 
Published: August 2011

SummaryThis paper discusses the problem of determining suitable roses of intersections for systems of planar thick fibres which cross and overlap. A planar Boolean model with long rectangular (or similar) grains is suggested as an appropriate mathematical model. It leads to a statistical estimator for the rose of intersections of the system of fibre spines. The method is used to analyse the microstructure of the outer layer of two samples of cardboard.
The observation of large magnetite (Fe3O4) crystals from magnetotactic bacteria by electron and atomic force microscopy
Authors: FARINA MARCOS , KACHAR BECHARA , LINS ULYSSES , BRODERICK RAYMOND , DE BARROS HENRIQUE LINS 
Published: August 2011

SUMMARYMagnetite crystals inside coccoid magnetotactic bacteria found in lagoons near Rio de Janeiro city were examined by electron microscopy (EM) and atomic force microscopy (AFM). For AFM, ultrathin sections of bacteria embedded in Epon resin were etched with an ethanolic NaOH solution and observed both in the height and in the force modes. Comparative electron microscope images were useful for identifying crystalline reliefs in the etched sections. Different situations representing particular arrangements of crystal chains were observed by AFM. The majority of the bacteria examined presented unusually large magnetite crystals which remained strongly attached in linear chains even after the laboratory procedures for their isolation. This behaviour is different from all other biogenic magnetite crystals isolated so far. It is suggested that this attachment is due to the strong field between individual crystals as well as to the contact areas, which are the largest observed until now. The correct identification of a particular topography by AFM as a crystal relief may be critical when crystals are not aligned in chains; in these cases the linear dimensions and the presence of well‐defined edges and faces are important features to be taken into account. Characterization of the crystal faces is important for the study of magnetotactic micro‐organisms since the crystalline habits seem to be species‐specific. Observation of etched sections proved to be a helpful approach for crystal relief observation, especially when small amounts of bacteria were available.
Quantitative analysis of variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) of cell/substrate contacts
Authors: BURMEISTER J. S. , TRUSKEY G. A. , REICHERT W. M. 
Published: August 2011

SUMMARYVariable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA‐TIRFM images are collected from well‐spread bovine aortic endothelial cells (BAEC) stained with a membrane‐bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm2) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm2).
A simple calibration for routine section thickness measurements using current density ratios
Authors: HENG Y. M. , OTTENSMEYER F. P. , ARSENAULT A. L. , SIMON G. T. 
Published: August 2011

SUMMARYA method to calibrate current density ratios for the determination of specimen thickness is presented. This method uses a tilt series from a single noncrystalline specimen to create different thicknesses; these are used to generate data points to establish the relationship between specimen thickness and current density ratio. The actual specimen thickness at 0° tilt was determined to an accuracy of 5 nm by a parallax method. From the calibration curves obtained, we observed that the current density ratio was sensitive to relative thickness changes on the same section of less than 1 nm when a 50‐μm objective aperture was used.
A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K
Authors: RICHTER K. 
Published: August 2011

SUMMARYMixtures of ethanol, 2‐propanol and 2‐butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning. Brought directly from room temperature to a cutting support at 140K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample. The mass hardens at lower temperatures fixing the sample well for microtomy. With ethanol: 2‐propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K.
The use of the stable isotope 44Ca in studies of calcium incorporation into dentin
Authors: LUNDGREN T. , ENGSTRÖM E. U. , LEVI‐SETTI R. , LINDE A. , NORÉN J. G. 
Published: August 2011

SUMMARYThe incorporation into rat incisor dentin of two calcium isotopes, the stable 44Ca and the radioactive 45Ca, was studied using secondary ion mass spectrometry (SIMS) stepscanning and imaging, and autoradiography, respectively. The results demonstrated a time‐dependent incorporation of the calcium isotopes into the mineral phase of dentin. With the SIMS step‐scanning, detecting 44Ca, the ion yield was high in the odontoblasts 2 min after intravenous injection. After 10 min a marked increase in signal intensity was found at the dentin mineralization front. This result was consistent with those obtained by 45Ca autoradiography; a peak of incorporation occurred 10 min after injection of the isotope. Likewise, localization of 44Ca to the mineralization front could be demonstrated 10 min after injection by SIMS imaging. In images obtained at earlier intervals, no such increase in ion yield could be detected. The results show that the nonradioactive, stable isotope 44Ca can be used as a marker for biomineralization in a similar way to radioactive 45Ca.
Minimizing sample evaporation in the environmental scanning electron microscope
Authors: CAMERON R. E. , DONALD A. M. 
Published: August 2011

SummaryThe ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.
Resolution in nonlinear laser scanning microscopy
Authors: DEITCHE J. , KEMPE M. , RUDOLPH W. 
Published: August 2011

SummaryThe lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single‐ or a two‐beam geometry is used, and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.
The influence of tissue processing on quantitative histopathology in breast cancer
Authors: LADEKARL M. 
Published: August 2011

SummaryObjective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume‐weighted mean volume of cancer cell nuclei, v̄V(nuc), was estimated using the method of point‐sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, āH(nuc), the nuclear volume fraction, VV(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained.The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5·0, 6·0, 7·0, 7·4 and 8·0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the v̄V(nuc) was 13% larger (2p = 0·004) and the mean ND 17% smaller (2p = 0·04) in hydroxyethyl‐methacrylate‐embedded samples from 17 tumours as compared to paraffin‐embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.
A novel application of microsphere perfusion and scanning electron microscopy to the identification of pulmonary arterioles in guinea‐pig and rabbit lungs
Authors: WALKER D. C. , HOSFORD S. , MACKENZIE A. 
Published: August 2011

SummaryIn arterioles of the lung the intravascular blood pressures are lower than in comparable vessels in the systemic circulation and the arteriole walls are thinner. Therefore, it is very difficult to distinguish between arterioles and venules of the same size using scanning electron microscopy. This study describes a novel application of latex microsphere perfusion and scanning electron microscopy which distinguishes between pulmonary arterioles and venules on the basis of endothelial cell morphology. Microspheres, 90 and 45 μm in diameter, were perfused into the arterial side of the pulmonary circulation of guinea‐pig and rabbit lungs. Scanning electron microscopy of the arterioles on both sides of the lodged microspheres indicated that the endothelial cells are spindle shaped. In contrast, the endothelial cells of equal diameter venules are polygonal. Furthermore, the nuclei of the arteriolar endothelial cells were significantly (P = 0·019) narrower than those of endothelial cells in venules of equal diameter. Finally, it was observed that the differences between arteriole and venule endothelial cells persisted distally to the capillaries.
Köhler illumination in the TEM: Fundamentals and advantages
Authors: BENNER G. , PROBST W. 
Published: August 2011

SummaryKöhler illumination is the most favourable design for the illumination path of an electron microscope with a condenser objective lens. The new illumination system of the EM 910 and EM 912 OMEGA allows both wide area (Köhler) illumination for TEM operation and spot illumination for analytical investigations. Compared to conventional systems and objective lenses with a condenser mini lens, this system offers many advantages. In addition to the homogeneous, highly coherent and parallel illumination of every point in the specimen, it offers advantages for selected area diffraction and spot scan mode.Combined with the electron optical selection of a condenser aperture, this illumination system provides the flexibility necessary to achieve optimum illumination for the specimen.
Optimal conditions for the use of correspondence analysis for element determination in EELS images
Authors: GELSEMA E. S. , BECKERS A. L. D. , DE BRUIJN W. C. 
Published: August 2011

SummaryCorrespondence analysis is used for the determination of element localization and element concentration in electron energy–loss imaging. The introduction of a new method for the quantification of element concentration embedded in the protocols of correspondence analysis may be regarded as a useful replacement of the power–law estimation technique, especially in regions of the spectrum where the latter method cannot be used. Conditions for the optimum use of the method are established.
Quantitative electron spectroscopic imaging in bio‐medicine: Methods for image acquisition, correction and analysis
Authors: BECKERS A. L. D. , DE BRUIJN W. C. , GELSEMA E. S. , CLETOM‐SOETEMAN M. I. , VAN EIJK H. G. 
Published: August 2011

SummaryMany questions about the metabolism of specific elements in the human body might be answered if elemental concentrations could be measured in situ in cells. With electron energy‐loss spectroscopic imaging (ESI), concentrations can potentially be determined with high spatial resolution. The theory of the quantification procedure has already been derived. Many practical instrument‐related problems, however, have to be solved. In the current research an energy‐filtering TEM is used and the image‐acquisition chain is examined in detail. Quantification requires images to be recorded over a large dynamic range. To solve this problem, the use of optical attenuation filters has been introduced. The use of the combination of a scintillator screen and a TV‐camera as a detection system has consequences for the processing of the data. Corrections for the camera photometric sensitivity and, to some extent, for shading are necessary. Further consequences of such a detection system for the correction of the element a‐specific spectral background and element detection are discussed. The derived methodology is tested in several ways and finally applied for the quantitative analysis of iron in liver parenchymal cells of a porphyria cutanea tarda patient.