Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).     

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.     

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.     

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.     

The dimensions and numbers of small vesicles in blood capillary endothelium in the hind legs of dogs, and their relation to vascular permeability

The dimensions and numbers of vesicles were determined in the blood capillary endothelium of the gastrocnemii muscle of dogs. These results permitted more accurate calculations of the number of vesicles crossing the endothelium in one direction/sec/(μm² (～6·2), and of the median vesicular attachment time (～8 sec). The probability of fusion occurring when a vesicle contacts a plasma membrane (α= 0·004) was unchanged: hence it was concluded, from the mean cellular width (0·21 μm) and the calculated cytoplasmic viscosity (～0·1 poise), that ～49% of the vesicles starting from one side reached the other one, and that their median transit time was ～1 sec.     

Assessment of collagen fibril spacing in relation to selected region of interest (ROI) on electron micrographs--application to the mammalian corneal stroma

AIMS: To evaluate measurements of collagen fibril spacing using different shaped regions of interest (ROI) on transmission electron micrograph (TEM) images of rabbit corneal stroma. METHODS: Following glutaraldehyde fixation and phosphotungstic acid staining, TEM images of collagen fibrils in cross section were projected at a final magnification close to 250,000 × to obtain overlays. Interfibril distances (IFDs; center‐to‐center spacing) were measured within different ROIs of the same nominal area (0.25 μm²) but different shape (with the length to width, L:W, ratio from 1:1 to 6:1). The IFD distribution was analyzed, and the 2D organization assessed using a radial distribution analysis. RESULTS: The fibrils had an average diameter of 35.3 ± 3.8 (SD) nm, packing density of 393 ± 4 fibrils / μm² and a fibril volume fraction of 0.39 ± 0.02. IFDs ranged from 29 to 1400 nm depending on the shape of the ROI, with average values ranging from 263 to 443 nm. By artificially selecting IFD data only to a radial distance of 250 nm, the average IFDs were just 145–157 nm. The radial distributions, to 250 nm, all showed a nearest neighbors first peak which shifted slightly from predominantly at 45–54 nm with more rectangular ROIs. The radial distribution profiles could be shown to be statistically different if the ROI L:W ratio was 2:1 or greater. CONCLUSION: Selection of an ROI for assessment of packing density and interfibril distances should be standardized for comparative assessments of TEMs of collagen fibrils. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Quantitative analysis of synapse structure with a semiautomatic interactive computer system: A study on identified pyramidal tract neurons in the sensory-motor cortex of cat

Pyramidal tract (Pt) neurons in the sensory-motor cortex of cat were labeled by injection of HRP into the spinal cord. Ultrastructural and quantitative analysis of the synaptic covering of their soma and apical dendrite (up to 100 μm from soma) was undertaken. We intercalated a visual-manual treatment between composite electron micrographs and a fully automated computer system and developed specific programs for evaluation of the morphometric data. Programs are included. A total of 412 synaptic boutons were examined that were found in contact with large Pt neurons. The mean linear percentage of the surface area covered by boutons was 26.2 ± 8.4% and the mean contacting length and cross-sectional area of the bouton profiles were 1.28 ± 0.58 μm and 0.89 ± 0.59 μm², respectively. All types of boutons with active zones accounted for 41.2% of the total. The distribution of two types of bouton (S- and F-type boutons, showing asymmetric and symmetric contacts, respectively) was examined quantitatively. The mean proportion of F-type boutons was 89.1% with a soma and S-type boutons contacting apical dendrites was 10.9%. In addition, GABAergic boutons were identified with the soma by immunocytochemistry with antibodies against glutamic acid decarboxylase. They formed symmetric synaptic contacts with the Pt cells that were identical to those formed by F-type boutons. The quantitative analysis revealed that synaptic clefts are narrower and synaptic vesicles are smaller in symmetric F-type boutons than in S-type boutons forming asymmetric contacts. These data establish that at least three parameters (postsynaptic density, synaptic cleft, and size of vesicles) can be utilized singly or in combination to identify GABAergic inhibitory synapses in neocortex.     

Tem Of Dislocations Under High Stress In Germanium And Doped Silicon

The stacking fault energies y of silicon (58 ± 6 mJ m^?2) and germanium (75 ± 10 mJ m^?2) were determined. Within the limits of accuracy γ was not found to change on doping with (13·8 mol m^?3 (8 × 10¹⁸ cm^?3) boron, and 1·17 mol m^?3 (7 × 10¹⁷ cm^?3) phosphorus). Freezing in dislocations under high shear stress reveals a different behaviour of screw dislocations: whereas these dislocations become wider in pure and p-silicon, they become narrower in n-silicon. From this we conclude the ratio of mobilities of the two 30° partials to be different in n- and p-silicon. Other observations on frozen dislocations are mentioned.     

Metallurgical interactions at the contacts GaAs interface

Interface morphology, phase composition, and elemental diffusion of Pt/Ti/Ge/Pd ohmic contacts to both n and p⁺-GaAs have been investigated as a function of annealing temperature. Structural and chemical results were correlated with specific contact resistances (ρ_c) measured for each thermal treatment. Annealing at 450°C yielded the lowest ρ_c, ～6.4 × 10^?7Ω-cm². The interface was observed to be smooth and abrupt. Two interface phases were detected; a primary phase, PdGe, and a secondary, Ga-rich Pd-Ga-As ternary phase. The presence of this ternary phase was found to be critical to contact formation on n-GaAs. The Ti and Pt layers remained pristine. Annealing at 550°C resulted in a slightly higher ρ_c, ～2.1 × 10^?6Ω-cm². There was significant elemental diffusion within the contact metal and minor diffusion into the GaAs substrate. The interface possessed limited areas of spiking with uniform composition. Annealing at 600°C proved to have a detrimental effect on the ρ_c, ～10^?4Ω-cm². This electrical degradation was accompanied by strong chemical intermixing between the contact and substrate, resulting in a continuous nonplanar interface with deep multiphase protrusions.     

Investigation of dislocation geometries in the diamond cubic structure

The weak-beam technique of electron microscopy (Cockayne, Ray & Whelan, 1969) has been applied to the examination of dislocations in germanium. These are shown to be dissociated into partial dislocations with a separation in the edge orientation of 5.5 ± 1·0 nm. A value for the stacking-fault energy of γ = 60 ± 8 mJ m^?2 (erg cm^?2) is deduced from the measured dissociation width as a function of orientation, using anisotropic elasticity theory.     

The determination of the 1/2<lll> {110} antiphase boundary energy of NiAl

A value for the 1/2 <111> {110} antiphase boundary (APB) energy in stoichiometric NiAl has been obtained by employing the weak-beam technique to resolve the separation of the two 1/2 <111> partial dislocations composing the <111 superdislocation. A separation of 4·5 nm is obtained corresponding to an APB energy of 200 ± 40 mJ m^?2 (erg cm²).     

Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion

The relaxation of fluorescence from diffraction‐limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo‐conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point‐spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D_ap. The model incorporates two novel features that make it of broad utility. First, the model includes the no‐flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D_ap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D_ap and η⁻¹ were obtained for the two proteins: for PAGFP, D_aq= 89 ± 2.4 μm² s⁻¹ (mean ± 95% confidence interval), and for EGFP D_aq= 91 ± 1.8 μm² s⁻¹. In CHO cells, the application of the model yielded D_ap= 20 ± 3 μm² s⁻¹ (PAGFP) and 19 ± 2 μm² s⁻¹ (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked ‘bound fraction’ explanation of the deviation of the terminal fluorescence recovery from its pre‐bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488‐nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.     

Microscopic evaluation of vesicles shed by erythrocytes at elevated temperatures

The images of human erythrocytes and vesicles were analyzed by a light microscopy system with spatial resolution of better than 90 nm. The samples were observed in an aqueous environment and required no freezing, dehydration, staining, shadowing, marking, or any other manipulation. Temperature elevation resulted in significant concentration increase of structurally transformed erythrocytes (echinocytes) and vesicles in the blood. The process of vesicle separation from spiculated erythrocytes was video recorded in real time. At a temperature of 37°C, mean vesicle concentrations and diameters were found to be 1.50 ± 0.35 × 10⁶ vesicles per microliter and 0.365 ± 0.065 μm, respectively. The vesicle concentration increased approximately threefold as the temperature increased from 37 to 40°C. It was estimated that 80% of all vesicles found in the blood are smaller than 0.4 μm. Accurate account of vesicle numbers and dimensions suggest that 86% of the lost erythrocyte material is lost not by vesiculation but by another, as yet, unknown mechanism. Microsc. Res. Tech. 76:1163–1170, 2013. © 2013 Wiley Periodicals, Inc.     

Study of solid particle erosion on AISI D2 using angular silicon carbide and steel round grit particles

Abstract

In this study, the performance of AISI D2 steel subjected to solid particle erosion tests was analysed. This material has applications for tools and dies for blanking, wood milling cutters, cold-extruding and other operations requiring high compressive strength and excellent wear resistance. The erosion tests performed by using a rig developed according to some parameters of the ASTM G76-95 standard. Two abrasive were used, angular silicon carbide (SiC) and steel round grit, both, with a particle size of 400–420 μm. This allowed comparing the erosion severity of each abrasive particle. The tests were conducted using four different incident angles 30, 45, 60 and 90° with a particle velocity of 24±2 m s^?1 and a flow rate of 21±2·5 g min^?1 for silicon carbide and 48·5±3·5 g min^?1 for the steel round grit. The exposure testing time was 10 min. Subsequently, the surface damage was analysed with a scanning electron microscope (SEM) to identify the wear mechanisms. Additionally, atomic force microscopy (AFM) was conducted in order to obtain roughness of the surface damage at 60°. The results indicated that higher amount of mass loss was obtained by angular silicon carbide particles.     

Local burn versus local cold induced acute effects on in vivo microcirculation and histomorphology of the human skin

Background: The impact of burns and colds on human skin microcirculation and histomorphology has not been compared as yet. Reflectance confocal microscopy (RCM) enables in vivo insight in human skin on cellular and subcellular levels. We evaluated analogies and differences of thermal injuries on microcirculation and histomorphology in vivo using RCM. Methods: Local superficial burn (6 female, 4 male; aged 28.4 ± 2.9 years, burn group) versus superficial cold (4 female, 6 male; aged 30.4 ± 5.2 years, cold group) was induced on the dorsum of the hand in an experimental immersion hand model. In vivo RCM was performed prior (control), immediately (t1) and 15 minutes (t2) following thermal injury to evaluate: Individual blood cell flow (IBCF), functional capillary density (FCD), epidermal thickness (ET), and granular cell size (GCS). Results: In the burn group, IBCF was increased at t1 (78.02 ± 2.60/min) and remained elevated at t2 (84.16 ± 3.04/min). In the cold group, IBCF decreased at t1 (12.62 ± 2.12 min) and increased at t2 (74.24 ± 3.14/min, P < 0.05) compared to the controls (58.23 ± 3.21/min). FCD was 6.74 ± 0.52/mm² in controls and increased at both t1 (7.82 ± 0.72/mm²) and t2 (8.02 ± 0.81/mm²) in the burn group. In the cold group, FCD decreased at t1 (2.60 ± 0.42/mm²) and increased at t2 (7.92 ± 0.44/mm², P < 0.05). ET increased at both t1 (43.12 ± 4.08 μm, P > 0.05) and t2 (47.26 ± 4.72 μm, P < 0.05) in the burn group. In the cold group, ET decreased at t1 (39.92 ± 3.14 μm, P > 0.05) and increased at t2 (44.72 ± 4.06 μm, P < 0.05) compared to the controls (41.26 ± 3.82 μm). Control GCS was 726.9 ± 59.4 μm² and increased at both t1 (739.8 ± 69.8 μm², P > 0.05) and t2 (762.6 ± 71.4 μm², P < 0.05) in the burn group. In the cold group, GCS decreased at t1 (712.4 ± 53.8 μm², P > 0.05) and increased at t2 (742.6 ± 64.8 μm², P < 0.05). Conclusions: Superficial burn induces more cellular destruction and cold leads to huge fluctuation in tissue perfusion, however, with moderate impact on histomorphology. The effect on dermal capillaries suggests a selective neural control and cold injuries might down‐regulate this system, much more than burns can activate it. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

An electron microprobe study of the influence of beam current density on the stability of detectable elements in mixed-salts (isoatomic) microdroplets

This paper shows that a means of accurately measuring beam current during microprobe analysis of inorganic fluid microdroplets is essential, since certain elements were sublimated from such specimens under easily achieved beam current densities, i.e. S at 1·8 nA/μm², K at 2·5 nA/μm², Na at 3·5 nA/μm², P at 5·3 nA/μm². In comparison, Cl was volatilized even under the mildest conditions used (0·35 nA/μm²), and Ca, Mg and Co were stable under the severest operating conditions (7·1 nA/μm²). Elements were less stable in large (3 μm diameter) droplets than in small (1 μm) droplets under identical irradiation conditions. The onset of volatilization is a direct function of the current delivered per unit area and not of the total integrated dose. The addition of 50 g/l of urea to the mixed-salts (isoatomic) solution, or (a) the mounting of the droplets so that the carbon-celloidin support film was interposed between them and the electron source, and (b) top-coating the droplets with carbon, did not, in general, raise the threshold of volatilization of a given element, but did effectively retard the rate of loss at current densities above the volatilization threshold. A literature survey confirmed that similar losses can occur from biological tissue specimens, albeit at higher beam current densities. Finally, the possibility that local specimen heating during electron/specimen interaction is a cause of element loss during microprobe analysis is discussed.     

Electron Microscope Studies Of Vpe Gainas Layer Structures Suitable For Use As Infrared Leds

Electron microscope investigations have been carried out on vapour grown (100) GaAs/GaInAs structures designed for use as infrared emitters of wavelength 1·06 μm. The structures consist of a GaAs substrate, a graded layer in which the indium concentration is increased from zero to 17 atomic %, and a constant composition Ga_0·83In_0·17As layer which contains a p-n junction. X-ray microprobe analysis of cross-sections of the slices established the uniformity of the grading. TEM analysis showed a dense and extensive asymmetric network of misfit dislocations (1 × 10¹² m^?2 (10⁸ cm^?2)) in the graded layer, threading dislocations and other anomalous contrast features extending from the graded layer through the p-n junction to the surface (local densities of 1 × 10¹¹–1 × 10¹² m^?2 (10⁷–10⁸ cm^?2)), and a planar network of dislocations just below the surface (spacing 0·2–2 μm). SEM EBIC and CL studies of the layer above the junction revealed dark spots, and a cross-grid of dark lines, which could be correlated with the threading defects, and the dislocation network just below the surface, respectively. The SEM results showed that these defects had a deleterious effect on the luminescent and electrical properties of the material in the vicinity of the p-n junction, and would therefore impair the performance of devices made from these layer structures.     

Interfacial energies and surface-tension forces involved in the preparation of thin,flat crystals of biological macromolecules for high-resolution electron microscopy

It is generally agreed that surface-tension forces and the direct interaction between the specimen and either the air-water interface or the water-substrate interface can influence significantly the preparation of biological materials for electron microscopy. Even so, there is relatively little systematic information available that would make it possible to control surface-tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm² or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose-embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air-water interface rather than adsorption of the specimen to the substrate. Surface-tension forces at the air-water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air-water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.     

Mechanical test and friction-mapping on recycled polypropylene beads using atomic force microscopy

Mechanical tests at sub-micron scales using force microscopy are often used for the characterization of materials. Here we report the mechanical, tribologic, and morphological characterization of recycled polypropylene beads using force spectroscopy and lateral-force microscopy. The compression-elastic moduli calculated using the Hertzian model for polypropylene beads was between 0.448 ± 0.010 and 1.044 ± 0.057 GPa. The grain size analysis revealed a significant correlation between the grain size and measured compression-elastic moduli. Friction-maps of recycled polypropylene beads obtained using lateral-force microscopy were also reported for 25 μm² scanning areas.