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Scanning acoustic microscopy (SAM), when applied to biological samples has the potential to resolve the longitudinal acoustic wave speed and hence stiffness of discrete tissue components. The heterogeneity of biological materials combined with the action of cryosectioning and rehydrating can, however, create variations in section topography. Here, we set out to determine how variations in specimen thickness influence apparent acoustic wave speed measurements
Cryosections (5μm nominal thickness) of human skin biopsies were adhered to glass slides before washing and rehydrating in water. Multiple regions (200x200 μm; n = 3) were imaged by SAM to generate acoustic wave speed maps. Subsequently co-localised 30x30 μm sub-regions were imaged by atomic force microscopy (AFM) in fluid. The images were then registered using Image J. Each pixel was allocated both a height and wave speed value before their relationship was then plotted on a scattergram. The mean section thickness measured by AFM was 3.48 ± 1.12 (SD) μm. Regional height variations influenced apparent wave speed measurements. A 3.5 μm height difference was associated with a 400 ms-1 increase in wave speed. In the present study we show that local variations in specimen thickness influence apparent wave speed. We also show that a true measure of wave speed can be calculated if the thickness of the specimen is known at each sampling point.
Graphene is a strong contender as a material to replace indium tin oxide as the transparent conductor of choice for electronic applications due to its exceptional electrical and optical properties. In this work, we present a study of graphene oxide (GO) films produced by inkjet-printing. The printed GO films are reduced using hydriodic acid (HI) and acetic acid vapour at low temperature. The reduced GO (rGO) films displayed good optical and electrical properties with a sheet resistance 6.8 kΩ/□ at a transmittance of 80%. In addition, we show that the conductivity of rGO films is related to both the size of individual GO sheets in the ink and the thickness of printed films. The rGO films using large size GO sheets displayed a thickness-independent conductivity of ∼ 4 × 104 S/m, while the rGO films using small size GO sheets showed a thickness-independent conductivity of ∼ 1.7 × 104 S/m. These properties are comparable to graphene films produced by solvent exfoliation. In summary, we demonstrate a scalable and potentially low-cost technique to produce rGO transparent films and a route to improve the conductivity of rGO films by controlling size of GO sheets in the ink.
In this study we have established a new approach to more accurately map
acoustic wave speed (which is a measure of stiffness) within soft biological
tissues at micrometer length scales using scanning acoustic microscopy. By
using thin (5 μm thick) histological sections of human skin and porcine
cartilage, this method exploits the phase information preserved in the
interference between acoustic waves reflected from the substrate surface as
well as internal reflections from the acoustic lens. A stack of images were
taken with the focus point of acoustic lens positioned at or above the
substrate surface, and processed pixel by pixel using custom software
developed with LABVIEW and IMAQ (National Instruments) to extract phase
information. Scanning parameters, such as acoustic wave frequency and gate
position were optimized to get reasonable phase and lateral resolution. The
contribution from substrate inclination or uneven scanning surface was
removed prior to further processing. The wave attenuation was also obtained
from these images.
The very high strengths that have been reported for nanoporous gold may be related to strain gradients within the deforming porous microstructure. We present a mechanism-based model for the strength of nanoporous foams that is derived from conventional models for the deformation of macroscopic foams and now includes the influence of strain gradients. This model predicts that the strength of the ligaments within the nanoporous gold is proportional to the ligament diameter raised to the power −0.5. We have used the model to analyze experimental data for the strength of nanoporous gold and find excellent agreement with published data.
In Type 1 and 2 diabetes tissue stiffening is evident from measurements of the gross mechanical properties of the vasculature. Elastic fibers play an important role in the mechanical function of vascular tissue, however, the effects of diabetes on individual elastic fiber components remains poorly defined. Fibrillin microfibrils, a key elastic fiber component, have a ‘beads-on-a-string’ structure with a periodicity of approximately 56 nm. We tested for possible disruption due to diabetes in fibrillin microfibrils isolated from rat aorta using an experimental model of Type 1 diabetes in rats. The isolated fibrillin microfibrils were imaged with atomic force microscopy (AFM) and image analysis techniques were used to characterise the microfibrils. Although there was no significant difference in mean microfibril length (control 23.2 repeats, SEM 6.2 repeats: diabetic 23.6 repeats, SEM 6.1 repeats: Mann Whitney U-test, p=0.391), mean periodicity was significantly reduced in microfibrils isolated from the diabetic rats (52.7 nm, SEM 0.4 nm) compared with age-matched controls (59.5 nm, SEM 0.4 nm) (p<0.001, Student's t-test). This study shows that diabetes leads to significant ultrastructural morphological changes in fibrillin microfibrils.
Drop on demand inkjet printing is a potential method for depositing enzymes onto electrodes for sensor applications. This technology offers drop sizes in the region of picolitres and allows a production rate up to 200 mm/s. This enables not only a more rapid method of device prototyping but also a method for possible miniaturization of the sensors themselves. However, previous work  has indicated that inkjet printing may cause a drop in the retained activity of the enzyme.
Here we assess the criticality of this drop in activity and how it may have been influenced by changes to the protein structure during printing. The enzyme used is glucose oxidase and the test methods include; protein analysis, in the form of analytical ultra-centrifugation and circular dichroism, scanning electron microscopy, atomic force microscopy and phase contrast microscopy, to analyse the surface topology of the electrodes and contact angle analysis, to assess the degree of spreading and the interactions between the drops and the electrode surface.
With glucose oxidase there is no change in the conformation, structure or hydrodynamic radius of the protein after printing. The analysis of the electrode surface shows a relatively smooth surface that is made up of individual graphite flakes laid down by a screen printing method. When contact angle and spreading analysis is carried out it demonstrates reliability in the printing process as well as a drop in the sessile volume of the drop in conjunction with a growth in the base diameter of the drop as expected. It also demonstrates a fairly quick rate of evaporation of the drop. Upon the addition of surfactants to the solution the spreading is seen to be more extensive in relation to the surfactant concentration, although some initial reduction in experienced at low concentrations which may be due to the absorption into the carbon surface.
The strength of submicron FCC structure metal columns, σ, fabricated by FIB machining or electrodeposition, shows a strong correlation with specimen diameter, d, with σ/μ = A(d/b)−0.63, where A is a constant, μ is the single crystal shear modulus resolved onto the slip system and b is the Burgers' vector. The strength of BCC structure metals does not show such a well defined correlation with size across different metals but the data occupies the same region of parameter space as with the FCC metals. Nanoporous gold specimens show a similar size-correlated behaviour but with an exponent of −0.5. This may indicate different mechanisms operating in each case.
Drop-on-demand inkjet printing is a fabrication technique that is capable of depositing materials layer-by-layer to form complex 3-dimensional (3-D) constructs. Here we present a new single drop delivery method in which both the matrix and cross-linker are present but separated through the use of vesicle packaging. Changing the printing parameters has little effect on the integrity of the calcium(II)-loaded vesicles, with calcium(II) released selectively by warming after printing. Alginate solutions containing calcium(II)-loaded vesicles were successfully printed and the printed layers were shown to gel on demand at 37 °C. The printed alginate layers were evaluated with regards to their potential to provide 3-D structures for cell culture.
We have carried out a TEM investigation of the micromechanisms of deformation in these nanoporous gold specimens after compression testing. We find that the nanoporous specimens show deformation localised to the nodes between the ligaments of the foamed structure, with very high densities of microtwins and Shockley partial dislocations in these regions. These deformation structures are very different from those seen after solid nanowires are tested in compression, which show very low dislocation densities and a few sparsely distributed twins. However, similar dislocation structures to those found in the nanoporous specimens are observed in the larger nanowires when they are deformed in bending. The currently accepted model for the deformation of nanoporous gold, implicitly assumes that the deformation of these structures is by bending near the nodes where ligaments intersect. We hypothesis that the much higher dislocation densities seen in both the nanoporous gold and the nanowires deformed in bending are evidence for the presence of geometrically necessary dislocations in these deformed structures.
Silver neodecanoate is sensitive to both ultra violet light (UV) and heat, and is a good inkjet printing precursor when dissolved in xylene. We have studied the electrical properties of inkjet printed silver samples, derived from silver neodecanoate ink, and investigated the influence of UV treatment before thermal curing the silver samples. In addition we have studied the influence of thermal pre-treatment on the printed samples. Thermally cured printed tracks and pads show minimum resistivity of approximately 3 x bulk silver. Their microstructure shows that the silver salt has converted to an interconnected network of silver nanoparticles after curing. The resistance of the printed tracks are shown to relate to the connectivity of the resulting sintered nanoparticle network as measured by the ratio of the sintered neck diameter to the original particle diameter.
We have measured the yield strength of gold nanowire forests with mean diameter 30, 60 and 70 nm fabricated by electro-deposition into porous alumina templates. All nanowire sizes showed yield strengths much greater than expected from polycrystalline gold specimens with the 30 nm specimens having a yield stress in excess of 1.4 GPa. We found no significant work hardening at plastic strains up to 30%. The strength of the nanowires as a function of wire diameter follows the same trend as has been reported for the compression strength of larger gold pillars reported in the literature. TEM observations of deformed wires are consistent with mechanisms of dislocation induced deformation.
The micro-mechanical properties of 5 μm thick histological sections of ferret aorta and vena cava were mapped as a function of distance from the outer adventitial layer using nanoindentation. In order to decouple the effect of the glass substrate on the elastic modulus of these thin sections, the nanoindentation data were analyzed using the extended Oliver and Pharr method which is readily accessible for coatings and layered materials with the software package, FilmDoctor®. In the aorta, the elastic modulus was found to decrease progressively from 35 MPa at the adventitia (outermost layer) to 8 MPa at the intima (innermost layer). This decrease in modulus was inversely correlated with elastic fibre density. In contrast, in the vena cava, the stiffest regions were found to be the adventitial (outer) and intimal (innermost) sections of the vessel cross-section. Both these regions were enriched in ECM components. The central region, thought to be largely cellular, had a relatively constant modulus of around 20 MPa. This study demonstrates that with this methodology it is possible to distinguish micro-mechanically between large arteries and veins, and therefore the same approach should allow age or disease related changes in the mechanical properties within a tissue to be quantified.
We have measured the yield strength of gold nanowires with diameters in the range from 30 to 70 nm fabricated by electro-deposition into porous alumina templates. All nanowire sizes showed yield strengths much greater than polycrystalline gold with the 30 nm specimens having a yield strength of 1.4 GPa. We found no significant work hardening at plastic strains up to 30%. The strength of the nanowires as a function of wire diameter follows the same trend as has been found for the compression strength of larger gold pillars reported in the literature. TEM observations of deformed wires are consistent with mechanisms of dislocation induced deformation. The strength of nanoporous gold nanowires measured by uniaxial compression test is also reported here. Although two different mechanisms are thought to operate in gold nanowires and nanoporous gold respectively, their strengths show very similar dependence on wire or ligament diameter. However the nanoporous material shows significant strain hardening.
Although the gross mechanical properties of ageing tissues have been extensively documented, biological tissues are highly heterogeneous and little is known concerning the variation of micro-mechanical properties within tissues. Here, we use Scanning Acoustic Microscopy (SAM) to map the acoustic wave speed (a measure of stiffness) as a function of distance from the outer adventitial layer of cryo-sectioned ferret aorta. With a 400 MHz lens, the images of the aorta samples matched those obtained following chemical fixation and staining of sections which were viewed with fluorescence microscopy. Quantitative analysis was conducted with a frequency scanning or V(f) technique by imaging the tissue from 960 MHz to 1.1 GHz. Undulating acoustic wave speed (stiffness) distributions corresponded with elastic fibre locations in the tissue; there was a decrease in wave speed of around 40 ms-1 from the adventitia (outer layer) to the intima (innermost).
Anodised aluminium oxide films have several applications in nanotechnology due to the ability to control the dimensions and ordering of the pores. Several techniques have been developed which allow for accurate control over the ordering of pore arrays. Examples of such techniques are the “Two-Step” method, and embossing with optical gratings or micro-machined Si templates. It will be demonstrated that it is possible to control the position of the pores using a nanoindenting device that has scratched the aluminium surface before anodising. The interpore distances accomplished via this technique were 200nm and 400nm by anodising in 0.3M (COOH)2 at 80V and 0.1M H3PO4 at 160V respectively. Hexagonal and square arrays were created by scratching the aluminium with a series of crosshatched lines.
The application of contemporary metallurgical knowledge and solid state bonding techniques provide for development of entirely new Mokumé Gane combinations. A multi-layered aluminium alloy is manufactured by the successive hot roll-bonding of two different aluminium alloys. The surface is then embossed with a selected pattern and machined back, exposing the various alloys. A range of suitable alloy combinations has been identified. When anodised, the surface presents the pattern as a strong contrast due to the differing anodising properties of the constituent alloy layers. The anodic coating can then be dyed with a wide range of colours to develop the decorative potential of the technique.
The interaction of an inkjet printed droplet with a substrate is of importance when determining the final size and shape of deposits. A simple model is proposed that relates droplet diameter, printed dot pitch and equilibrium contact angle to as-printed track width. Reasonable agreement was found between the model and final track width, with slight over-prediction accounted for by removal of the organic component of the ink during heat treatment. Immediately after droplet impact, the drop may spread to a considerably greater extent than predicted by equilibrium because of the kinetic energy of the droplet in flight. Simulations of droplet impact showed that the maximum droplet spread decreased linearly with equilibrium contact angle. Recoil of these droplets towards their equilibrium shape occurred above a threshold contact angle. This threshold was greater than expected, suggesting an energy barrier preventing recoil or the kinetics of recoil being too slow for recoil to occur within the timeframe of this study.
A piezoelectric drop on demand printer has been used to print primary human osteoblast and bovine chondrocyte cells. After deposition the cells were incubated at 37°C and characterised using optical microscopy, SEM and cell viability assays. Cells showed a robust response to printing exhibiting signs of proliferation and spreading. Increasing the drop velocity results in a reduced cell survival and proliferation rates but both cell types grew to confluence after printing under all conditions studied.