Imaging textural variation in the acoustoelastic coefficient of aluminum using surface acoustic waves.

Much interest has arisen in nonlinear acoustic techniques because of their reported sensitivity to variations in residual stress, fatigue life, and creep damage when compared to traditional linear ultrasonic techniques. However, there is also evidence that the nonlinear acoustic properties are also sensitive to material microstructure. As many industrially relevant materials have a polycrystalline structure, this could potentially complicate the monitoring of material processes when using nonlinear acoustics. Variations in the nonlinear acoustoelastic coefficient on the same length scale as the microstructure of a polycrystalline sample of aluminum are investigated in this paper. This is achieved by the development of a measurement protocol that allows imaging of the acoustoelastic response of a material across a samples surface at the same time as imaging the microstructure. The development, validation, and limitations of this technique are discussed. The nonlinear acoustic response is found to vary spatially by a large factor (>20) between different grains. A relationship is observed when the spatial variation of the acoustoelastic coefficient is compared to the variation in material microstructure.

Determination of the acoustoelastic coefficient for surface acoustic waves using dynamic acoustoelastography: an alternative to static strain.

The third-order elastic constants of a material are believed to be sensitive to residual stress, fatigue, and creep damage. The acoustoelastic coefficient is directly related to these third-order elastic constants. Several techniques have been developed to monitor the acoustoelastic coefficient using ultrasound. In this article, two techniques to impose stress on a sample are compared, one using the classical method of applying a static strain using a bending jig and the other applying a dynamic stress due to the presence of an acoustic wave. Results on aluminum samples are compared. Both techniques are found to produce similar values for the acoustoelastic coefficient. The dynamic strain technique however has the advantages that it can be applied to large, real world components, in situ, while ensuring the measurement takes place in the nondestructive, elastic regime.

Spatial modulation microscopy for real-time imaging of plasmonic nanoparticles and cells.

Spatial modulation microscopy (SMM) is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the SMM technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticles.

Surface plasmon microscopy: resolution, sensitivity and crosstalk.

This paper develops the theoretical framework to understand the capability of the interferometric surface plasmon microscope to quantify sample properties in a confined region. We use rigorous diffraction theory to quantify the ability of the system to measure local properties and eliminate crosstalk from adjacent regions. We argue that the interferometric system in the defocused condition defines the measured point of excitation and reradiation of the surface plasmons; which greatly improves localisation. We also present results for the noninterferometric microscope, which confirm that the interferometric based system can perform quantitative measurements over smaller regions.

Double-grating-structured light microscopy using plasmonic nanoparticle arrays.

Structured illumination increases the spatial bandwidth of optical microscopes. We demonstrate bandwidth extension using a physical grating placed close to the sample. This comprises an array of elongated nanoparticles, whose localized surface plasmon resonance is polarization dependent. By arranging the particle orientation to vary with position the grating can be moved by changing the input polarization. A projected optical grating provides an additional independent mechanism for bandwidth extension. Experimental results showing bandwidth improvement in one direction are presented, and the measures necessary to extend the technique for routine imaging are discussed.

Rapid photoreflectance spectroscopy for strained silicon metrology.

We present an improved photoreflectance (PR) spectroscopy technique upon the prior art in providing a rapid acquisition method of the PR spectrum in a simultaneous and multiplexed manner. Rapid PR (RPR) application is the on-line monitoring of strained silicon. Shrinkage in the silicon bandgap is measured and converted to strain, using theoretical models. Experimental RPR results are in good correlation with Raman spectroscopy.

Studying protein binding to conjugated gold nanospheres; application of Mie light scattering to reaction kinetics.

The study of protein interactions is an area of much interest, particularly towards obtaining more detailed information about biological processes. Current methods involve the use of complicated, specialised techniques which are beyond the scope of most laboratories. Here, we show how information about the binding of proteins to conjugated gold nanospheres can be obtained using straightforward experimental techniques. A Perkin Elmer LS 55 luminescence spectrometer was used to observe the changes in light scattering caused by the binding of complementary proteins to conjugated nanoparticles, measured by the intensity change over time. Mie theory simulations have been used to predict the expected observations and to quantify the changes in intensity as a function of surface coverage. Further kinetic studies have been carried out at 530 nm to obtain more detailed information about the processes involved in the binding reaction. Thus, we have demonstrated that the interaction of proteins can be studied using a straightforward method which provides information about surface coverage and reaction kinetics.

Measurement of elastic nonlinearity using remote laser ultrasonics and CHeap Optical Transducers and dual frequency surface acoustic waves.

A nonlinear ultrasonic technique for evaluating material elastic nonlinearity has been developed. It measures the phase modulation of a high frequency (82MHz) surface acoustic wave interacting with a low frequency (1MHz) high amplitude stress inducing surface acoustic wave. A new breed of optical transducers has been developed and used for the generation and detection of the high frequency wave. The CHeap Optical Transducer (CHOT) is an ultrasonic transducer system, optically activated and read by a laser. We show that CHOTs offer advantages over alternative transducers. CHOTs and nonlinear ultrasonics have great potential for aerospace applications. Results measuring changes in ultrasonic velocity corresponding to different stress states of the sample are presented on fused silica and aluminium.

Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy.

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles.

This paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to real-time imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly-L-lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron-sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.

Surface plasmon-assisted widefield non-linear imaging of gold structures.

In this paper, we demonstrate how the confinement and enhancement of optical fields by surface plasmon resonance can allow non-linear microscopy to be performed in a simple, cost-effective widefield configuration, rather than the more usual laser-scanning arrangement. We present second harmonic and two-photon luminescence widefield images of dielectric and gold samples obtained with both prism-based and high numerical aperture objective ('prismless') microscope arrangements.

Imaging performance of widefield solid immersion lens microscopy.

We investigate the performance of a widefield imaging system employing an aplanatic solid immersion lens. Off-axis imaging quality is examined theoretically at different radii and thicknesses of the aplanatic solid immersion lens. It is found that field curvature is the major aberration affecting the imaging quality. Aberrations are measured experimentally, and the results are in very good agreement with those obtained from simulations and demonstrate the situations where high quality images can be obtained with the aplanatic solid immersion lens.

Aberrations in materials with random inhomogeneities.

Materials that consist of a random microstructure can affect ultrasonic measurements--reducing signal strength, increasing noise, and reducing measurement accuracy--through scattering and aberration of the acoustic field. To account for these adverse effects a phase screen model, alongside the stochastic wave equation, has been developed. This approach allows the field and study aberrations to be modeled from a statistical point of view. Experimental evidence of aberration and statistical properties of the measured acoustic field are shown. A measured correlation function of the acoustic field is interlinked to mean crystallite size by using a theoretical coherence function that can be mainly described by the correlation length and wave velocity variation of microstructure. The estimation of the mean crystallite size using this technique would provide some insight into material characterization.

Two-photon fluorescence surface wave microscopy.

This paper demonstrates the principle of two-photon surface wave microscopy with a view to applications on biological samples. We describe a modified scanning optical microscope, which uses specially prepared coverslips. These coverslips are designed to support the propagation of surface waves capable of large field enhancements. We also discuss the beam conditioning necessary to ensure efficient use of the available illumination. Two-photon surface wave fluorescent excitation is demonstrated on fluorescent nanospheres, demonstrating a point spread function width of approximately 220 nm at an illumination wavelength of 925 nm. The potential of non-linear surface wave excitation for both fluorescence and harmonic imaging microscopy is discussed.

High-resolution wide-field surface plasmon microscopy.

This paper describes the application of a Köhler illuminated high-resolution wide-field microscope using surface plasmons to provide the image contrast. The response of the microscope to a grating structure in both the Fourier and the image planes is presented to demonstrate image formation by surface waves. The effect of spatial filtering in the back focal (Fourier) plane to enhance image contrast is described. We also discuss how the surface wave contrast mechanism affects the imaging performance of the microscope and discuss factors that can be expected to lead to even greater improvements in lateral resolution and sensitivity.

Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator.

This paper presents a brief account of a novel optical microscope, which combines the advantages of two well-known techniques, namely phase contrast and phase stepping, to provide high contrast imaging and precision measurements. The inclusion of a programmable liquid crystal spatial light modulator provides for the phase stepping required, while also allowing flexibility for future improvements. The results shown reveal an important aspect of the system to facilitate quantitative sample measurements, with an enhancement of optical resolution compared with conventional optical imaging systems.

Surface plasmon fluorescence microscopy: an analysis.

This paper examines the imaging performance of surface plasmon microscopes operating in the far field. Until recently the accepted view has been that the achievable lateral resolution with a surface plasmon imaging system is limited by propagation decay length of the surface plasmons rather than by diffraction. We show by simulation that the lateral resolution of a surface plasmon imaging system can be comparable to that of the best optical microscopes. We also show that new imaging modes such as two-photon and also second harmonic surface plasmon microscopy are extremely promising imaging modes that may be expected to be powerful techniques for the analysis of surface structures.

Optical V(z) for high-resolution 2pi surface plasmon microscopy.

Surface plasmons are electromagnetic surface waves whose k vectors are greater than that of free-space radiation. We excite surface plasmons by using an oil-immersion lens, which forms one arm of an interferometer. We demonstrate the way in which the characteristic output variation with defocus is determined by the propagation properties of the surface plasmons, which leads to diffraction-limited surface plasmon microscopy in the far field.

Fast, all-optical Rayleigh wave microscope: imaging on isotropic and anisotropic materials.

A fast, non-contact Rayleigh wave scanning microscope is demonstrated, which is capable of scan rates of up to a maximum of 1000 measurements/s with typical speeds of up to 250 measurements/s 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 (CGH). The generating laser produces around 30 pulses at 82 MHz and additional harmonics at 164 and 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 specialized knife-edge detector. The microscope has been developed for imaging on isotropic materials. Despite this, the system can be used on anisotropic materials, but imaging and interpreting images can be difficult. The anisotropy and grain structure of the material can distort the Rayleigh wavefront, leading to signal loss. A model has been developed to simulate polycrystalline-anisotropic materials; this is discussed along with possible solutions that would overcome the problems associated with anisotropy. Rayleigh wave amplitude images are demonstrated on silicon nitride at 82 and 164 MHz and on polycrystalline aluminium at 82 MHz.

High-resolution scanning surface-plasmon microscopy.

Surface plasmons (SP's) are electromagnetic surface waves that propagate along the interface between conductors and dielectrics. The k vector of these waves is larger than the free-space wave vector. The importance of SP's lies in the fact that they are extremely sensitive to small changes in the dielectric properties of substances that are in contact with the conductors. This property means that SP's have many sensor applications; however, when they are used in microscopic applications the lateral resolution is limited to several micrometers. We discuss how this limit can be overcome by use of defocused high-numerical-aperture liquid-immersion objectives. We also present SP images that demonstrate a resolution comparable with that expected from high-numerical-aperture optical microscopes. Finally, we discuss how ultrahigh-numerical-aperture objectives with numerical apertures greater than 1.5 can be expected to have considerable influence on biological imaging.