Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

Atomic force microscopy of human hair

The atomic force microscope (AFM) was used to investigate the surface architecture of the entire lengths of cleaned human head hairs. Many features previously seen with the scanning electron microscope (SEM) were identified. However, the AFM has provided much greater detail and, in particular, the hair's cuticular surfaces appear not to be as smooth as had been previously supposed. A consistent feature was of step discontinuities or "ghosts" on the scale surfaces. These delineated the original location of each overlying scale before its edge had been chipped away. There was a change in the longitudinal angular presentation of the surfaces about each ghost. This means the distal ends of each cuticle cell have been synthesised in the follicle to be thicker than where that same cuticle cell is bounded on both sides by other cuticle cells. The undamaged outer cuticular surfaces at the root end of each hair were covered everywhere by longitudinal ridges (striations). Where the hair surface was worn, the striations terminated at a scale edge ghost. The ridges were approximately 9 nm high and were in parallel array with a lateral repeat spacing of about 350 nm. The striations are evidently formed on the outer surface of each cuticle cell following earlier contact in the hair follicle with the inner root sheath. The study of stained transverse sections of hairs in the transmission electron microscope (TEM) is suggested as a means for throwing some light on the underlying structure and chemistry of the striations. Finally, our AFM studies have revealed that the surface of the freshly emergent hair gradually changes over a distance of about 20 mm and that the surface of the hair for most of its length is quite different from that near the root. This is likely to be of import to those engaged in the hair toiletries industry.     

Investigation of dyed human hair fibres using apertureless near-field scanning optical microscopy

We present the first studies of dyed human hair fibres performed with an apertureless scanning near‐field optical microscope. Samples consisted of 5‐µm‐thick cross‐sections, the hair fibres being bleached and then dyed before being cut. Hair dyed with two molecular probes diffusing deep inside the fibre or mainly spreading at its periphery were investigated at a wavelength of 655 nm. An optical resolution of about 50 nm was achieved, well below the diffraction limit; the images exhibited different optical contrasts in the cuticle region, depending on the nature of the dye. Our results suggest that the dye that remains confined at the hair periphery is mainly located at its surface and in the endocuticle.     

Specific labelling of thiol groups in mammalian keratin suitable for electron microscope studies

Quantitative Polarographic studies of the reaction between methylmercuric iodide and variously reduced wool keratin indicate that reaction occurs specifically with the thiol groups from cystine. Since loss of protein from fibres is negligible, this organomercurial is preferred as a selective amino acid label in electron microscope studies. Similar reactions of wool with parachloromercuribenzoate (PCMB) result in extensive structural modifications. The mercury mercaptides were shown to be thermally unstable in the electron beam and cooling of the specimen between — 140°C and — 88°C coupled with low beam intensities was found necessary to study the original distribution of the mercurials in the samples. Since a direct relationship between the electron scattering sites of the mercurial and cystine within the keratin was established, it has been possible to demonstrate unambiguously for the first time that: (a) There exists a radial distribution of cystine within cuticular cells. Indeed a new sulphur-rich component at the outer edge of the ‘a’ layer in peripheral cuticular cells has been identified. (b) Para-cortical cells of intact keratin contain a higher proportion of cystine than ortho-type cells. (c) A dense concentration of cystine labelled by the electron scattering centres (mercurials) corresponds closely to an intermicrofibrillar-matrix system in the cortex. Furthermore, a method has been devised for determining the cystine content of the exocuticle using data obtained from electron micrographs.     

Returning to the same area of hair surfaces before and after treatment: a longitudinal AFM technique

We report the use of longitudinal (aspect ratio > 1 : 1) scanning atomic force microscopy as an aid in returning to the same area of hair fibres after bleaching, treatment with a commercial shampoo or the application of a ‘leave‐on’ conditioner product. The bleaching treatment used in this study was not found to affect the cuticular architecture and lateral force microscopy (LFM) also showed little difference after treatment, reflecting the homogeneity of the newly revealed surfaces. After treatment with a commercial shampoo, the hair sample again showed very little difference in topography or lateral force characteristics. Hair treated with the leave‐on conditioner product also showed no major topographical changes. LFM traces, however, showed regions between the ghost edge, marking the original position of the scale edge before cuticular erosion, and the existing scale edge, to have higher frictional properties than distal regions of the cuticle. A thin film of the leave‐on product thus seems to form in this region and extends from the foot of the scale edge.     

Fluorescence anisotropy near-field scanning optical microscopy (FANSOM): a new technique for nanoscale microviscometry

A near-field scanning optical microscope system was implemented and adapted for nanoscale steady-state fluorescence anisotropy measurement. The system as implemented can resolve 0.1 cP microviscosity variations with a resolution of 250 nm laterally in the near field, or 10 μm when employed in a vertical scanning mode. The system was initially used to investigate the extent of microviscous vicinal water over surfaces of varying hydrophilicity. Water above a cleaved mica surface was found to have a decreased microviscosity, while water above a hydrophobic surface showed no change (detection limit 0.1 cP at 30+ nm from the surface).     

Novel MMC microstructure with tailored distribution of the reinforcing phase

Saffil short fibre agglomerates with diameters of 0.4 mm to 1 mm have been prepared using a tumbling technique. These were packed and infiltrated with molten 6061 Al alloy to make a metal matrix composite (MMC) with a novel microstructure in which the composite spheres are randomly distributed in the fibre-free aluminium matrix. In parallel, a commercial preform made of identical Saffil alumina short fibres and having the same fibre volume faction was used to prepare a conventional MMC by the same technique. Microstructural observation indicates that, within the composite spheres, the local volume fraction of fibre decreases from the outer layer to the centre region.
The energy absorption during fracture was estimated by using a three point loading test on notched samples and was compared with that for conventional MMCs. Preliminary results suggest that this novel MMC possesses higher energy absorption capability and hence better damage tolerance. The fracture surfaces were examined by scanning electron microscopy in order to inform these experimental results.     

Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

Charge effect in point projection images of carbon fibres

Nanometre-sized carbon fibres across holes have been observed in a lensless point projection field-emission microscope operating between 100 and 300 eV. At sufficiently high magnification fringe patterns appear; with the help of simulations we show that they are strongly dependent on the charge density of the fibres. These patterns are characterized by an odd number of fringes with a central fringe that becomes very bright as the charge increases. Average diameter and linear charge density have been obtained with remarkable precision from analysis of fringes. Charge distribution from the middle to the edge of fibres has been investigated as well as narrowings at localized places on the fringe pattern. From these two examples, the limits of the models used for the simulations and those of the data acquisition system are discussed. Finally, this work emphasizes the fact that the fringe pattern masks the actual form of the fibre and that it is necessary to take account of the charge effect to interpret this diffraction pattern.     

Scanning probe microscopy of biological samples and other surfaces

Scanning probe microscopes derived from the scanning tunnelling microscope (STM) offer new ways to examine surfaces of biological samples and technologically important materials. The surfaces of conductive and semiconductive samples can readily be imaged with the STM. Unfortunately, most surfaces are not conductive. Three alternative approaches were used in our laboratory to image such surfaces. 1. Crystals of an amino acid were imaged with the atomic force microscope (AFM) to molecular resolution with a force of order 10^?8 N. However, it appears that for most biological systems to be imaged, the atomic force microscope should be able to operate at forces at least one and perhaps several orders of magnitude smaller. The substitution of optical detection of the cantilever bending for the measurement by electron tunnelling improved the reliability of the instrument considerably. 2. Conductive replicas of non-conductive surfaces enabled the imaging of biological surfaces with an STM with a lateral resolution comparable to that of the transmission electron microscope. Unlike the transmission electron microscope, the STM also measures the heights of the features. 3. The scanning ion conductance microscope scans a micropipette with an opening diameter of 0·04-0·1 μm at constant ionic conductance over a surface covered with a conducting solution (e.g., the surface of plant leaves in saline solution).     

Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi-quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass-like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

Optical coherence tomography as a tool for characterization of complex biological surfaces

The advent of scanning electron microscopy has facilitated our understanding of the biology in relation to surface microstructure of many invertebrates. In recent years, interest in biomimetics and bio‐inspired materials has further propelled the search for novel microstructures from natural surfaces. As this search widens in diversity to nurture deeper understanding of form and function, the need often arises to examine rare specimens. Unfortunately, most methods for characterization of the microtopography of natural surfaces are sacrificial, and as such, place limiting constraints on research progress in situations where only a few rare specimens are known, such as the rich resources lodged in natural history museum collections. In this paper, we introduce the use of optical coherence tomography (OCT) as a noninvasive tool for bioimaging surface microtopography of crab shells. The technique enables the capture of microstructures down to micron level using low coherence near‐infrared light source. OCT has allowed surface microtopography imaging on crab shells to be carried out rapidly and in a nondestructive manner, compared to the scanning electron microscope technique. The microtopography of four preserved crab specimens from Acanthodromia margarita, Ranina ranina, Conchoecetes intermedius and Dromia dormia imaged using OCT were similar to images obtained from scanning electron microscope, showing that OCT imaging retains the overall morphological form during the scanning process. By comparing the physical lengths of the spinal structures from images obtained from OCT and scanning electron microscope, the results showed that dimensional integrity of the images captured from OCT was also maintained.     

High voltage electron microscopy of the central nervous system in Golgi preparations

Elastic tissue has been identified in the scanning electron microscope by two independent methods. In one case specifically stained fibres were examined by both light and scanning electron microscopy and in the other chemically purified elastic fibres from ligamentum nuchae were studied. Elastic fibres above 2 μm diameter were found to have a central amorphous core and an irregularly corrugated and undulating outer surface. This model is in good agreement with that proposed on the basis of previous transmission electron microscopic studies.     

Behavior of alloy Ti–6Al–4V under pre-fretting and subsequent fatigue conditions

The mechanical behavior and microstructural changes in Ti–6Al–4V were determined in fretting tests, followed by axial fatigue tests. Prior to fatigue testing, specimens were subjected to fretting conditions over a range of contact stresses and fretting displacements. Fretting frequency was 100 Hz. High cycle fatigue (HCF) tests were run at 1000 Hz. The fretting test involved a flat-on-flat, bare Ti–6Al–4V/bare Ti–6Al–4V fretting system. The fretting process typically generated very shallow surface cracks at the ends of the wear scar. Subsequently, these shallow cracks were observed to propagate in axial fatigue tests, reducing the fatigue life significantly. Evidence of frictional heating during fretting was observed in the formation of scale-like oxide in the wear scar. Formation of oxides appeared to increase with increasing contact stress. Increased oxygen content was detected in the near surface regions of specimens. Large near surface deformation was typically observed within the wear scar. The contact geometry and slight tilting of the stationary fretting pad influenced the character of the fretting scar and the fretting-induced cracking. Fracture surfaces exhibited featureless, battered surfaces at the crack origins followed by (a) cleavage-type crack propagation, (b) formation of fatigue striations, and (c) final ductile tearing.     

Investigations on the leaf anatomy and ultrastructure of grapevine (Vitis vinifera) under heat stress

Leaf anatomical and ultrastructural responses of "Razegui" and "Muscat Italia" grapevine cultivars to high temperatures were studied under controlled conditions (T > 36°C), based on photonic and electron microscopy. Histological studies performed on leaves from heat-stressed and control grapevines revealed thicker leaf blades under high temperature conditions. Environmental scanning electron microscopy of leaf surfaces from both cultivars allowed observing sinuate epidermal cells on the leaves of grapevines cultivated under heat stress and irregular giant oblong pores on their adaxial surface. When observed by transmission electron microscopy, leaf cross sections in grapevines cultivated under high temperature conditions exhibited folded cuticle and cell wall on the adaxial epidermis layer. Therefore, significantly greater cell wall thicknesses were measured under heat stress than control conditions in both cultivars. Regarding chloroplasts, they were more globular in shape under heat stress compared with control conditions and had disorganized thylakoids with a reduced thickness of grana stacking. The size of starch granule decreased, while the number of plastoglobules increased with heat stress, indicating a reduced carbon metabolism and a beginning of senescence within the 3-month heat stress period. This study confirms widespread adaptive properties in two grapevine cultivars in response to high temperature stress.     

Calibration of AFM cantilever stiffness: a microfabricated array of reflective springs

Calibration of the spring constant of atomic force microscope (AFM) cantilevers is necessary for the measurement of nanonewton and piconewton forces, which are critical to analytical applications of AFM in the analysis of polymer surfaces, biological structures and organic molecules.

We have developed a compact and easy-to-use reference standard for this calibration. The new artifact consists of an array of 12 dual spiral-cantilever springs, each supporting a mirrored polycrystalline silicon disc of 160 μm in diameter. These devices were fabricated by a three-layer polysilicon surface micromachining method, including a reflective layer of gold on chromium. We call such an array a Microfabricated Array of Reference Springs (MARS). These devices have a number of advantages. Cantilever calibration using this device is straightforward and rapid. The devices have very small inertia, and are therefore resistant to shock and vibration. This means they need no careful treatment except reasonably clean laboratory conditions.

The array spans the range of spring constant from around 0.16 to 11 N/m important in AFM, allowing almost all contact-mode AFM cantilevers to be calibrated easily and rapidly. Each device incorporates its own discrete gold mirror to improve reflectivity. The incorporation of a gold mirror both simplifies calibration of the devices themselves (via Doppler velocimetry) and allows interferometric calibration of the AFM z-axis using the apparent periodicity in the force–distance curve before contact. Therefore, from a single force–distance curve, taking about one second to acquire, one can calibrate the cantilever spring constant and, optionally, the z-axis scale. These are all the data one needs to make accurate and reliable force measurements.     

A versatile multipurpose scanning probe microscope

A combined scanning probe microscope has been developed that allows simultaneous operation as a non‐contact/tapping mode atomic force microscope, a scattering near‐field optical microscope, and a scanning tunnelling microscope on conductive samples. The instrument is based on a commercial optical microscope. It operates with etched tungsten tips and exploits a tuning fork detection system for tip/sample distance control. The system has been tested on a p‐doped silicon substrate with aluminium depositions, being able to discriminate the two materials by the electrical and optical images with a lateral resolution of 130 nm.     

Towards phonon photonics: scattering-type near-field optical microscopy reveals phonon-enhanced near-field interaction

Diffraction limits the spatial resolution in classical microscopy or the dimensions of optical circuits to about half the illumination wavelength. Scanning near-field microscopy can overcome this limitation by exploiting the evanescent near fields existing close to any illuminated object. We use a scattering-type near-field optical microscope (s-SNOM) that uses the illuminated metal tip of an atomic force microscope (AFM) to act as scattering near-field probe. The presented images are direct evidence that the s-SNOM enables optical imaging at a spatial resolution on a 10 nm scale, independent of the wavelength used (λ=633 nm and 10 μm). Operating the microscope at specific mid-infrared frequencies we found a tip-induced phonon-polariton resonance on flat polar crystals such as SiC and Si₃N₄. Being a spectral fingerprint of any polar material such phonon-enhanced near-field interaction has enormous applicability in nondestructive, material-specific infrared microscopy at nanoscale resolution. The potential of s-SNOM to study eigenfields of surface polaritons in nanostructures opens the door to the development of phonon photonics—a proposed infrared nanotechnology that uses localized or propagating surface phonon polaritons for probing, manipulating and guiding infrared light in nanoscale devices, analogous to plasmon photonics.     

Rapid cryofixation of rabbit muscle fibres after a temperature jump

We describe a procedure whereby structural changes that occur in muscle fibres after a rapid temperature jump can be captured by cryofixation. In the thick filament from rabbit and other mammalian skeletal muscles there is a rapid transition from a non‐helical to a helical structure as the temperature is raised from 273 K towards physiological levels. This transition is accompanied by characteristic intensity changes in the X‐ray diffraction pattern of the muscle. In our experiments to capture these changes, single fibres of glycerinated psoas muscle were subjected to a Joule temperature jump of 15–30 K from ~278 K in air. We have developed a freezing method using a modified Gatan cryosnapper in which a pair of liquid nitrogen‐cooled copper jaws were projected under pressure and closed on the fibre between 50 and 100 ms after the temperature jump. The frozen fibres were freeze‐substituted and embedded for electron microscopy. Transverse and longitudinal sections of relaxed ‘cold’ (~278 K) and temperature‐jumped fibres as well as rigor fibres were obtained. Fourier transforms of the images from the three preparations showed differences in the relative intensities of the reflections from the hexagonal filament lattice and in those of the helix‐based layer lines, similar to the differences seen by X‐ray diffraction. We conclude that we have preserved the ‘hot’ structure and that cryofixation is sufficiently fast to prevent the transition back to the ‘cold’ state.