Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014. © 2013 Wiley Periodicals, Inc.     

Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

Introduction: The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Methods: Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. Results: In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Conclusion: Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

3D‐quantum interferometer using silicon microring‐embedded gold grating circuit

A 3D (three‐dimensional) quantum interferometer consisting of a silicon microring circuit proposed. The interferometer based on the electron spin cloud projections generated by microring‐embedded gold grating. The electron cloud oscillations result from the excitation of the gold grating at the center of the silicon microring by the dark soliton pulse of 1.50 μm center wavelength. The electron cloud spin‐down, spin‐up automatically formed in the two axes (x, y, respectively) and propagated along the z‐axis. In this proposal, the sensing mechanism of the circuit is manipulated by varying the reflector gold lengths of the sensing arm. The electron cloud spin coupled and changed by changing the gold lengths. The sensitivity measurement of the 3D quantum interferometer for three gold layer lengths of 100 nm, 500 nm, and 1,000 nm is (47.62 nm fs^?1, ±0.4762 fs^?1, ±0.01 nm^?1), (238.10 nm fs^?1, ±0.4762 fs^?1, ±0.002 nm^?1), (476.20 nm fs^?1, ±0.4762 fs^?1, ±0.001 nm^?1), respectively. The used circuit parameters are the real ones that can be fabricated by the currently available technology. Moreover, the silicon micro ring circuit acts as a plasmonic antenna, which can apply for wireless quantum communication. The electron cloud spin projection space–time control can apply for quantum cellular automata.     

Tensile and Friction Properties of Tin Bronze Matrix Composites Reinforced by Carbon Fibers

Copper-coated carbon fibers and carbon fiber (CF)-reinforced tin bronze matrix composites were prepared by chemical plating and powder metallurgy, respectively. Copper-coated CFs were characterized with field emission scanning electron microscope. The effects of CF volume fraction on the friction properties and tensile properties of the composites were investigated. The results showed that the composites exhibited lower friction coefficient and higher tensile strength than tin bronze 6-6-3 with the chemical composition of Cu-6 wt%Sn-6%Zn-3%Pb. The friction coefficient of the composites decreased with the increasing of the CF volume fraction. The composite with 9 vol.% CFs showed the highest hardness and tensile strength, which were, respectively, about 1.8× and 1.68× higher than those of the tin bronze 6-6-3.     

Three-dimensional morphometrical study of dendritic spines of the granule cell in the rat dentate gyrus with HVEM stereo images

Number, length, and diameters of dendritic spines of the granule cell in the dorsal leaf of the rat dentate gyrus were measured by using high-voltage electron microscope stereo images of 5-m?m-thick Golgi preparations with the aid of a three-dimensional image analyzer system. Spine densities of 2.02 ± 0.28, 2.28 ± 0.33, and 3.36± 0.35 per 1 μm at distal, middle, and proximal portions of the dendrite were obtained. These values were about 1.6-fold of the previous light microscopical report. Mean three-dimensional spine length were 1.244 ± 0.506 μm, 1.262 ± 0.563 μm, and 1.254 ± 0.584 μm at distal, middle, and proximal portions, respectively, which were about 1.4 times longer than those measured in two dimensions. By using measured morphometrical parameters of spines such as lengths, diameters, and population densities, total spine surface areas of 2.401 μm², 2.806 μm², and 4.180 μm² per 1 μm of the dendrite at distal, middle, and proximal portions, respectively, were obtained. The total surface area of dendrite was about doubled by the addition of the spines at each dendritic portion. The advantageous features and the problems of the present method are discussed.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Development of a new analytical electron microscopy technique to quantify the chemistry of planar defects and to measure accurately solute segregation to grain boundaries

A new technique of analytical transmission electron microscopy called ConceptEM has been developed for determining highly accurately small amounts of solute or dopant atoms incorporated into well‐defined planar defects such as stacking faults, grain boundaries or interfaces. The method is based on recording series of analytical spectra taken with different electron beam diameters on the same position centred above a defect that is orientated either edge‐on or slightly inclined with respect to the electron beam. It can be applied to energy‐dispersive X‐ray spectroscopy or electron energy‐loss spectroscopy and necessitates only a nano‐probe modus but no scanning unit. Reliability and accuracy have been tested numerically under various conditions using simulations for a specific geometry, as a function of specimen thickness, material, acceleration voltage, collection angle, random beam displacements and solid solubility. The accuracy has been found to be substantially better (by factors of 5–10) than that of any other current standard technique based on single measurements. Our calculations suggest an accuracy in the determination of the Gibbsian solute excess at a special grain boundary down to ±1% of a monolayer, i.e. around ±0.1 atoms nm^?2 under typical experimental conditions, with a maximum error about twice as large. The parameter limiting a straightforward analysis is found to be the solid solubility, which itself, however, can be measured accurately by the technique so that it can be taken into account quantitatively and the above‐stated precision is retained.     

Wide field scanning slit in vivo confocal microscopy of flattening-induced corneal bands and ridges

We used a wide field scanning slit confocal microscope to examine the response of the in vivo human cornea to flattening. Flattening-induced effects consisted of (1) anterior corneal mosaic, which appeared as a meshwork of intersecting stromal and Bowman's layer bands with overlying epithelial ridges; (2) deep and middle stromal bands, which were narrower than and unrelated in position to the anterior corneal mosaic; and (3) posterior surface ridges. The posterior surface ridges projected posteriorly into the anterior chamber consisted of endothelium, Descemet's membrane, and posterior stroma, and were unrelated in position to posterior stromal bands. Confocal microscopy is a promising modality in the examination of the cornea and its response to mechanical stress.     

Advanced light microscopy core facilities: Balancing service,science and career

Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463–479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.     

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10³ e/nm²) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10¹⁰ e/nm²) is required resulting in mass loss that may be attributable to ‘knock-on’ radiation damage.     

Analysis of directly frozen macromolecules and tissues in the field-emission STEM

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (? 10³ e/nm²) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm². For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm² regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.     

Quantification of metallic nanoparticle morphology on TiO2 using HAADF-STEM tomography

Electron tomography is applied to photocatalytic gold/titanium oxide and gold/silver/titanium oxide samples. In order to obtain a tilt series for the electron tomography measurement, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) is used under cryogenic conditions. Dedicated programs have been developed for measuring volume, surface area, thickness distribution and nearest-neighbour distance of metallic nanoparticles on samples. Using these quantification programs, the 3D morphology of gold and silver nanoparticles is accurately characterized. We paid particular attention to the quantitative measurement of surface area. The measurement error of the method and appropriate magnification are defined using spherical nanoparticle models. We measured the 3D morphology of gold nanoparticles supported on titanium oxide (total volume=6.5×10⁵ [nm³], surface area=1.4×10⁵ [nm²], and average nearest-neighbour distance=40 [nm]).     

Performance of plastic gear made of carbon fiber reinforced poly-ether-ether-ketone: Part 2

Five kinds of PEEK/CF composites, made by blending poly-ether-ether-ketone (PEEK) with three kinds of PAN type carbon fibers (CFs) and two kinds of pitch type CFs respectively, were injection-molded into gears. Their gear performance such as the load capability and the wear of tooth were evaluated. The wear properties of PEEK/CF gears extremely varied depending on the kinds of CFs, when the same type gears were combined and grease was initially applied. Also, the load capabilities were significantly influenced by the affinity between PEEK and CF. A composite gear reinforced with CF of the highest density indicated the highest load capability irrespective of the test conditions, due to the lowest abrasive property of the CF as well as the excellent affinity between PEEK and CF. Its load capability under a high temperature running condition was found to be superior to that of polyamideimide (PAI) composite gear or polyphenylenesulphide (PPS) composite gear.     

Iodine potassium iodide improves the contrast‐to‐noise ratio of micro‐computed tomography images of the human middle ear

High‐resolution imaging of middle‐ear geometry is necessary for finite‐element modeling. Although micro‐computed tomography (microCT) is widely used because of its ability to image bony structures of the middle ear, it is difficult to visualize soft tissues – including the tympanic membrane and the suspensory ligaments/tendons – because of lack of contrast. The objective of this research is to quantitatively evaluate the efficacy of iodine potassium iodide (IKI) solution as a contrast agent. Six human temporal bones were used in this experiment, which were obtained in right‐left pairs, from three cadaveric heads. All bones were fixed using formaldehyde. Three bones (one from each pair) were stained in IKI solution for 2 days, whereas the other three were not stained. Samples were scanned using a microCT system at a resolution of 20 μm. Eight soft tissues in the middle ear were segmented: anterior mallear ligament, incudomallear joint, lateral mallear ligament, posterior incudal ligament, stapedial annular ligament, stapedius muscle, tympanic membrane and tensor tympani muscle. Contrast‐to‐noise ratios (CNRs) of each soft tissue were calculated for each temporal bone. Combined CNRs of the soft tissues in unstained samples were 6.1 ± 3.0, whereas they were 8.1 ± 2.7 in stained samples. Results from Welch's t‐test indicate significant difference between the two groups at a 95% confidence interval. Results for paired t‐tests for each of the individual soft tissues also indicated significant improvement of contrast in all tissues after staining. Relatively large soft tissues in the middle ear such as the tympanic membrane and the tensor tympani muscle were impacted by staining more than smaller tissues such as the stapedial annular ligament. The increase in contrast with IKI solution confirms its potential application in automatic segmentation of the middle‐ear soft tissues.     

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 10³ e/nm² limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.     

The preparation of field-ion-microscope specimens containing grain boundaries

A back polishing technique has been devised which increases the efficiency of a field-ion-microscope study of grain boundaries. The rate of back polishing is predicted from the relationship d_md = 2·5 × 10⁴ nm²/msec. This relationship is shown to be valid experimentally and theoretically. Its validity implies the persistence of constant current density conditions during electropolishing of sharp, needle-shaped specimens down to diameters of 100 nm.     

A study on residual fatigue bending strength and damage behavior of CFRP composites subjected to impact loadings

This paper evaluates the static and fatigue bending strengths of CFRP (carbon-fiber reinforced plastic) laminates having impact damages, e.g., foreign object damages (FOD). Composite laminates used in this experiment are CF/EPOXY and CF/PEEK orthotropy laminated plates with two-interfaces [0°₄/90°₄]_s A steel ball launched by an air gun collides against the CFRP laminates to generate impact damages. The damage growth during a bending fatigue test is observed by a scanning acoustic microscope (SAM). When the impacted side is compressed, the residual fatigue bending strength of CF/PEEK specimen P is greater than that of CF/EPOXY specimen B. On the other hand, when the impacted side is in tension, the residual fatigue bending strength of CF/PEEK specimen P is smaller than that of CF/EPOXY specimen B. In the case of impacted-side compression, the fracture is propagated from the transverse crack generated near the impact point. In the case of impacted-side tension, however, the fracture develops toward the impact point from the edge of interface-B delamination.     

Study of structures at the boundary and defects in organic thin films of perchlorocoronene by high-resolution and analytical transmission electron microscopy

Both the periodic and non-periodic structures of perchlorocoronene (C₂₄Cl₁₂) crystals were characterized by high-resolution transmission electron microscopy (HRTEM), electron diffraction (ED), electron energy-loss spectroscopy (EELS), and energy-filtered transmission electron microscopy (EFTEM). The HRTEM images at the boundary of the C₂₄Cl₁₂ crystals exhibit the flexibility of defect structures, where molecules align to compensate for the discontinuity between two different domains. Emphasized by the filtered images, it was found that the non-periodic regions are created everywhere with a small electron beam irradiation (∼10⁶ electrons nm⁻²) and then spread over the entire regions to completely destroy the periodic structures after a higher electron dose (∼2×10⁶ electrons nm⁻²). The effect of the electron beam irradiation was monitored by ED, EELS, and EFTEM, where periodic structures and content elements are well preserved up to 10⁶ electrons nm⁻², but chlorine atoms decreased with a much higher electron dose. This is explained by the breakage of the C–Cl bond to detach chlorine atoms, confirmed by energy-loss near the edge structures (ELNES) of carbon π? peaks and chlorine loss at the edge of the specimen, as well as by theoretical simulation. The detachment of chlorine is localized at the peripheral edge around a hole confirmed by core-loss EFTEM imaging.     

A convenient method for electron tomography sample preparation using a focused ion beam

Here we report a new sample preparation method for three‐dimensional electron tomography. The method uses the standard film deposition and focused ion beam (FIB) methods to significantly reduce the problems arising from the projected sample thickness at high tilt angles. The method can be used to prepare tomography samples that can be imaged up to a ±75° tilt range which is sufficient for many practical applications. The method can minimize the problem of Ga⁺ contamination, as compared to the case of FIB preparation of rod‐shaped samples, and provides extended thin regions for standard 2D projection analyses. Microsc. Res. Tech. 75:1165–1169, 2012. © 2012 Wiley Periodicals, Inc.     

Observations of human corneal epithelium by tandem scanning confocal microscope

We applied the tandem scanning confocal microscope (TSCM) to 30 healthy human corneas of 3 normal volunteers and 27 patients with cataract and retinal detachment to observe normal corneal epithelial cells in vivo. All corneas were normal under slit lamp microscopic examination. The superficial and basal epithelial cells close to the basal lamina in the central cornea were recorded on videotape and analyzed by a computer-assisted digitizer. The mean cell areas of superficial cells exposed at the surface and basal cells at the horizontal section were 624 ± 109 μm² and 66 ± 5 μm², respectively. The ratio of superficial to basal mean cell area was 11.0 ± 4.5. TSCM was thus useful in evaluating the relationship between superficial and basal cells in human corneal epithelium in vivo.