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Cell culture is essential to many areas of biology ranging from the fundamental study of cell biology to the application of cells for therapeutic purposes in Regenerative Medicine. Common to all these areas is the need to characterise cell populations under culture. Currently, cell populations are routinely monitored using conventional biological analysis e.g. cell surface markers, gene expression. This approach is destructive, not suitable for in-process measurements and renders time course experiments impossible. Alternatively non-destructive approaches that assess cell morphology can also be used, with light microscopy techniques (e.g. bright field, phase contrast imaging) being the primary methods. These microscopy techniques can sometimes be combined with the use of exogenous labels such as fluorescent markers. This can provide functional information but has the disadvantage that such cell modifications are invasive and potentially toxic to the cells.

Surface plasmon (SP) microscopy offers great potential for bioimaging applications particularly for quantitative measurement of refractive index over localized regions. Our aim is to measure variations in refractive index so that important events such as antibody-antigen binding can be measured on smaller spatial scales than is possible with conventional prism based SP imaging. To this end we have used rigorous coupled wave analysis (RCWA) to develop a theoretical framework to understand the capability of the SP microscope to measure local changes in refractive index. Our results show that the non-interferometric microscope configuration gives results which are strongly dependent on the propagation length of the SPs, whereas in the interference mode the measured path of the SPs is determined by the sample defocus and SPs following different paths are not detected. This gives better localization of the measurement of local refractive index as well as a more predictable value as it depends on the optical set rather than sample properties.

Epithelial tissues form the barrier between different fluid compartments throughout the body, lining and delineating the borders between tissues to control homeostasis and provide for the functions of secretion, absorption, and volume regulation. Key to these physiological roles is the transepithelial transport of fluid and solutes. Vectorial transport is possible due to the highly polarised cytoarchitecture, with different ion transporters and other proteins present in the apical vs basolateral membrane domains, which are separated by “tight junctions” which ring the lateral membranes of each cell. These separate the apical and basolateral compartments, providing intercellular adhesion and controlling permeation via the intercellular pathway. By varying the composition and function of the pools of transport proteins between these segregated membrane domains, directed transport is achieved thus promoting homeostasis. The retinal pigment epithelium (RPE) lies behind the neural retina, forming the blood-retinal barrier and providing for the homeostasitic and biochemical support of the photoreceptors and other neuronal layers. Much is known of RPE physiology at the macroscopic level since dysregulation or pathology have profound consequences for the visual system. Lacking is the detailed knowledge of biophysical mechanisms and local intercompartmental dynamics by which fluid and solute transport is achieved and regulated at the cellular and subcellular level.

We have been actively developing surface plasmon (SP) microscopy as a technique which has great potential for measuring localized changes in refractive index. We have used various interference techniques1 to demonstrate the potential of the method as have other researchers2. The so called V(z) response gives an indication properties of the SPs generated on the sample.

Widefield interferometry is a useful technique for the fast visualisation of low contrast objects. In addition, the spatial resolution of images is improved and quantitative measurement becomes possible with this technique.

Structured illumination microscopy (SIM) using grating excitation can be used to extend the bandwidth of fluorescent microscopy by approximately a factor of 2 in the linear regime. If some of the fluorescent molecules are saturated even greater improvements in resolution are possible; this may, however, lead to high levels of photobleaching and phototoxicity. In this paper we present preliminary results that show a simple grating structure separated by a propagation region (see figure 1) can improve the resolution by a far greater factor than this offering the opportunity for resolution close to 50nm. Our present results are proof of concept results on relatively low numerical aperture systems. The potential for the higher lateral resolution relies on the fact that (i) the grating structure does not depend on the illumination optics and can thus be finer than possible with a grating formed by illumination through the lens and (ii) the propagation region can be made from a material with high refractive index is possible with immersion oils.

The impact of Alzheimer's disease (AD) is projected to become one of the major challenges to modern society: over the next few years the incidence of AD will reach 1 million people in the UK alone with an estimated annual economic cost of £30 billion. Unfortunately, the existing diagnostic procedures are inadequate for early disease detection and do not always differentiate AD from other dementias. In addition, they are time consuming, expensive and of limited availability outside specialist centres. Definitive AD diagnosis is still only available post-mortem and the development of enhanced diagnostic strategies are, therefore, highly desirable. We have identified a panel of AD biomarkers incorporating proteins whose diagnostic power arises from their interrelated patterns and we are developing an instrument for point-of-care diagnostic tests that can be used in clinical settings (community medical centres and hospitals) as a screening tool for these markers. Availability of a reliable biomarker test for AD would increase the accuracy of disease diagnosis, aid earlier disease detection including automation for community screening and allow for improved treatment targeting. Whilst no cure for AD is currently available, earlier, more accurate and cost-effective detection of AD will be an important component for facilitating development and implementation of newer, disease modifying therapies (amyloid vaccines). Our approach would also allow expensive resources, particularly emerging state-of-the-art imaging technologies, to be more effectively utilised

The use of optical gratings for the generation and detection of ultrahigh frequency narrowband surface acoustic waves is well known. Detection of surface acoustic waves (SAWs) using an optical grating, (without an interferometric set-up) relies on diffraction of light from the grating. Cheap Optical Transducers (CHOTs) for SAWs are optical ultrasonic transducers which are designed to optimise the diffraction from such gratings to yield a local interferometer. They have been demonstrated for typical NonDestructive evaluation (NDE) frequencies (0.5 and 100 MHz). In this paper, we discuss how the CHOTs operate and how their detection mechanism breaks down, when the optical diffraction orders from the grating become evanescent upon moving to multi GHz frequency range. We show that it is possible to design devices with enhanced sensitivity, in this range. This is done by adding a thin background film underneath grating fingers, in order to optimise and exploit the interaction between the deformation of the grating and the evanescent fields/ resonances involved in such gratings. The result is a novel operating mechanism, based on an energy balance between the 0-order reflection and resistive heating. These novel transducers are called evanescent wave CHOT (eCHOT). We have used Finite Element Method (FEM) modelling to link the physical displacements caused by the elastic waves with the optical behaviour. We demonstrated that the devices show enhanced sensitivity over a wider range of parameters, than possible with a device designed with conventional CHOT specifications, making the eCHOT an ideal candidate for GHz / nano scale ultrasonics. The eCHOT involves highly resistive thin films and yields thin structures advantageous for ultrahigh frequency ultrasonics. The operating mechanism of the eCHOT suggests that more exotic structures could be built to enhance the sensitivity of these devices.

Materials with random microstructure can have an adverse effect on ultrasonic measurements through scattering and aberration of the acoustic field. We have developed a theoretical and experimental technique for characterising the effect of the material microstructure on the propagation of acoustic waves. The theoretical method predicts propagation of the statistical properties in the random medium. Using this model and a novel experimental technique we can extract materials properties from observations of acoustic aberrations. The microstructure has been modelled as having grains randomly orientated and weakly anisotropic. Moreover, individuals grains are treated to be equiaxed. This is summarised by assuming that the correlation function for the wave number has a Gaussian shape. Under this assumption, the approximated power correlation function for the acoustic field has been obtained using the stochastic wave equation for random media, along with numerical simulation using phase screen theory. The experimental evidence that aberrations are frequency dependent is presented. Multiple c- scans on titanium were performed using a 10MHz transducer as an ultrasonic source. The output of the transducer gives substantial frequency components between 6MHz and 16MHz. The variation of the frequency on a fixed sample enables us to examine different measurement regimes (higher frequencies correspond to large grains). The statistical analysis and estimated power correlation function from measurements are compared to a modelled power correlation function. The correlation length and standard deviation of the wave number define the power correlation function of the field. This function has been fitted to the estimated power correlation from measurements on titanium 6-4, and values for the variance and correlation length were obtained. The paper demonstrate that the stochastic model is capable of quantitative prediction of the predominant wave scattering effect in granular materials. of acoustic waves will be random. The grain microstructure may have various effects on ultrasonic wave propagation. It attenuates, scatter and aberrates ultrasonic waves as they propagate within this medium. Generally speaking aberrations are wavefront distortion of ultrasonic waves propagating in random medium. It would be desirable to quantify the strength of aberrations on a material with random microstructure. The main observation relies on the transverse coherence of the acoustic field as it propagates. The first step is to measure aberrations then the strength by means of transverse power correlation function of the acoustic field, which will be the subject of forthcoming sections. The theoretical description of SAW propagation is based on a scalar stochastic theory for waves in a random medium. Therefore it is possible to obtain an expression for second order moments of the field which carries information of microstructure. The behaviour of power correlation is mainly dictated by the scale of the inhomogeneity which is a rough estimation of grain size. The theoretical derivation is not included in this paper as the main purposes is to show how this technique behaves under different regimes of frequency and also because it has been developed in a previous paper, (3).

The make-up of the material microstructure of multi-grained materials such as titanium alloys and aluminum is of great interest to many in industries such as aerospace. The ability to map the material microstructure - in effect to image the grains - quickly and in a nondestructive manner would be useful from both a process control perspective and in the area of nondestructive evaluation. There are several techniques in the field that are capable of imaging grain structure, including simple etching, orientation imaging microscopy and scanning acoustic microscopy; all have their strengths and weaknesses. We present a new ultrasonic technique that can directly and quantitatively image the local surface acoustic wave (SAW) velocity over the surface of a material. Material microstructure can be determined if the phase velocity of the grains varies with grain orientation. The acoustic waves are generated and detected by lasers and as well as being noncontact, the technique is relatively fast, can cope with large samples, and is totally nondestructive. The new technique involves varying the spatial parameters of the excitation pattern in real time to maximize the generation efficiency of the surface acoustic waves at the phase velocity of the material, in the region of excitation. By repeating this over the sample surface, a surface wave phase velocity map can be produced. As well as describing the velocity mapping technique in detail, several example results of industrially-relevant materials acquired using our new instrument are presented. The limit to the quantitative lateral resolution of the instrument is discussed, and how this relates to the qualitative lateral resolution. Results indicating the practical limit of the accuracy of velocity measure- ments are also presented. We demonstrate imaging on several samples (40mm 2 )a nd different materials. This shows that the the instrument can reveal the underlying microstructure and image areas of anomalous grain structure that may be significant for the performance of the material. The instrument currently works on smooth samples but can be extended to work on rough surfaces and components with unprepared surfaces and consequently this has high industrial relevance.

The present work investigates the capabilities of a novel widefield surface plasmon microscope (WSPR) and its potential applications in biology. Here in particular, we concentrate on imaging interfacial interactions between specific antibodies and the proteins laminin and fibronectin, both patterned onto prefabricated Au/Cr/glass substrates. These experiments are used to demonstrate the high sensitivity, good contrast and high lateral resolution (close to a micron) of the new system. These results indicate that our microscope may form the basis for a new generation of fast assay systems.

Many common engineering materials can aberrate high frequency acoustic waves. The source of the aberrations is spatial velocity variations resulting from the material microstructure. Aberrations can degrade acoustic measurements by distorting the acoustic wavefront and causing acoustic speckle. A new technique is demonstrated which optimises the spatial distribution of the generation of the ultrasound to compensate for acoustic aberrations. The technique uses a Green's function for the material which is experimentally determined. In aberrating materials the Green's function is a function of source and observation position, G(R,S). The spatial coherence is optimised in the required measurement region to suppress acoustic speckle. The technique is used to improve the accuracy of velocity measurements in steels with high frequency SAWs. The technique was performed using the adaptive optical scanning acoustic microscope (AOSAM) at the University of Nottingham, UK.

A noncontact and completely damage-free instrument capable of rapid high resolution vector contrast imaging using surface acoustic waves is described. The laser source of the ultrasound generates around 30 pulses at 82MHz and harmonics thereof, and it is possible to image at several discrete frequencies-82MHz, 164MHz, 246MHz and above-simultaneously. Adaption of the spatial distribution of the excitation source to accommodate a wide range of materials and circumstances is trivial, since a spatial light modulator is used to produce an arbitrary distribution of incident optical energy. This is used to good effect in anisotropic materials with random grain structures, since the material microstructure perturbs the surface waves as they propagate on the material surface. The wavefront aberrations are detected by an acoustic wavefront sensor, and the excitation profile is adjusted to correct for the aberrations. This increases the accuracy and reliability of the measurements, and can increase the usable frequency range or propagation distance for a given material. The adaptable source enables the instrument to be used for purposes other than linear surface wave detection and c-scan imaging; for instance, the SAW phase velocity of anisotropic crystals may be determined by wavenumber spectroscopy achieved by adapting the source k-vector and finding the peak in excitation. Also, there is considerable interest in nonlinear and harmonic detection and imaging, and the adaptable source may be used to suppress the harmonics naturally present in the excitation source. This is important for harmonic imaging, where it is necessary to ensure that the relatively weak harmonics generated by the nonlinear material properties are not swamped by those present in the source.

This paper discusses how laser ultrasonic imaging technology may be used for defect detection. The poor single to noise ratio and the consequent potential for damage to the sample have hindered the widespread application of laser ultrasound. We discuss how our approach overcomes many of these problems. Moreover, we discuss the specific and potentially important advantages that arise from the frequency flexibility, absence of couplant and the ability to control the generated wavefront.

The authors propose a method of interpreting amplitude only V(z) curves, derivable from ray theory without arbitrary assumptions. The method has been applied to V(z) curves measured with both cylindrical and spherical lenses, achieving an absolute accuracy of Rayleigh velocity of better than one part in one thousand in each case. This level of accuracy matches that previously available only from cylindrical lenses. It is shown how optimal filtering of the V(z) curve may be performed to eliminate high-frequency coherent ripple due to internal lens reverberations and to minimize noise with minimum degradation of the actual curve. In numerical simulations the method has been shown to have a high degree of immunity from noise and to be effective for short ranges of defocuses. The processing algorithm has no arbitrary steps and should be easily capable of hands-off automation. >

Laser generated ultrasound has many attractive properties. This paper discusses two methods of controlling the distribution of optical energy on the sample by the use of an acousto-optic cell and the use of computer generated holograms. These methods may be used to enhance the generation of harmonics of the fundamental frequency of the ultrasound. It is possible with this technique to reduce the peak optical intensity on the sample without decreasing the amplitude of the ultrasound. The acousto-optic cell technique splits a single generating source into two separated by a controllable distance. By varying the separation of the sources different harmonics can be enhanced or suppressed. Computer generated holograms may be used to generate complex optical distributions which can be used to focus ultrasound and enhance high frequency harmonics. Furthermore, holograms may be designed to focus the light onto a curved surface allowing examination of samples with difficult geometries. Experimental results are presented which demonstrate these principles.

A fast non-contact Rayleigh wave scanning microscope is demonstrated. This is capable of scan rates of up to a maximum of 1000 measurements per second with typical speeds in excess of 40 measurements per second on real samples. The system uses a mode-locked, Q-switched Nd:YAG laser operating at a mode-locked frequency of 82 MHz, and a Q-switch frequency of 1 kHz. The Q-switch frequency determines the upper limit of the scanning rate. The generating laser illumination is delivered and controlled by a computer generated hologram. The generating laser produces around 30 pulses at 82 MHz, and additional harmonics at 164 MHz, 246 MHz and above. The microscope can operate at these harmonics provided the spatial bandwidth of the optics and the temporal bandwidth of the electronics are suitable. The ultrasound is detected with a specialised knife-edge detector. This technique is very successful on isotropic materials. At present imaging and interpreting images taken using this technique can be difficult on anisotropic materials because the anisotropy can distort the Rayleigh wavefront leading to signal loss. Rayleigh wave amplitude images are demonstrated on silicon nitride at 82 MHz, and 164 MHz and aluminium at 82 MHz.

Digital image correlation has been used to measure microscopic deformation in thermally stressed microelectronics devices. Displacement precisions of better than 0.03 pixels have been achieved by combining nonintegral pixel shifting of subimages and artificial neural networks (ANNs). The ANNs are trained to estimate the subpixel element of the object displacement from the digital correlation. Although similar accuracies can be obtained by curve-fitting to the correlation peaks and differentiating, the neural approach has the advantage that it allows fast subpixel. displacement analysis over a range of object textures without knowledge of the analytical form of the correlation peaks.

In this work we evaluate the applicability of two nondestructive thermal wave techniques for ion implant monitoring, these being photodisplacement and photothermal radiometric microscopy. Experimental results are presented for BFz implants into single-crystal (100) silicon wafers with implantation parameters varied as follows: Ion dose (lE12-3E15 ions/cm* ) and ion energy (50-200 keV). Using these results, the sensitivity of the above techniques to material parameters such as lattice damage, ion penetration depth and surface recombination velocity is discussed. We will show that while no one technique is applicable to all implant characteristics, the various techniques examined are complementary and thus application specific.

We briefly describe how one may calculate the response of a cylindrical lens when imaging over a specimen with lateral discontinuities by use of a Green's function for the propagation of Rayleigh waves. We then present the expected contrast from various features. Finally we show how the ideas arising from the theory can aid interpretion and understanding of acoustic images.

This paper presents experimental surface acoustic wave velocity dispersion data, obtained from thin film tungsten silicide layers (500-2500 A) cosputtered on single-crystal silicon wafers, as a function of various technologically relevant thermal processing treatments. The velocity dispersion data was determined using an acoustic microscope operating at 375 MHz via the acoustic material signature (AMs) technique. Transmission electron microscopy and X-ray diffraction analysis were used to characterise the microstructure of these thin films. Simple mathematical expressions for the transmission of a surface wave along a varying polycrystalline structure were then used to interpret the experimental data, the approximate model thus developed enables us to access the interface conditions between grains in the material.