Ultra-high-resolution optical imaging for silicon integrated-circuit inspection

This thesis concerns the development of novel resolution-enhancing optical techniques for the purposes of non-destructive sub-surface semiconductor integrated-circuit (IC) inspection. This was achieved by utilising solid immersion lens (SIL) technology, polarisation-dependent imaging, pupil-function engineering and optical coherence tomography (OCT). A SIL-enhanced two-photon optical beam induced current (TOBIC) microscope was constructed for the acquisition of ultra-high-resolution two- and three-dimensional images of a silicon flip-chip using a 1.55μm modelocked Er:fibre laser. This technology provided diffraction-limited lateral and axial resolutions of 166nm and 100nm, respectively - an order of magnitude improvement over previous TOBIC imaging work. The ultra-high numerical aperture (NA) provided by SIL-imaging in silicon (NA=3.5) was used to show, for the first time, the presence of polarisation-dependent vectorialfield effects in an image. These effects were modelled using vector diffraction theory to confirm the increasing ellipticity of the focal-plane energy density distribution as the NA of the system approaches unity. An unprecedented resolution performance ranging from 240nm to ~100nm was obtained, depending of the state of polarisation used. The resolution-enhancing effects of pupil-function engineering were investigated and implemented into a nonlinear polarisation-dependent SIL-enhanced laser microscope to demonstrate a minimum resolution performance of 70nm in a silicon flip-chip. The performance of the annular apertures used in this work was modelled using vectorial diffraction theory to interpret the experimentally-obtained images. The development of an ultra-high-resolution high-dynamic-range OCT system is reported which utilised a broadband supercontinuum source and a balanced-detection scheme in a time-domain Michelson interferometer to achieve an axial resolution of 2.5μm (in air). The examination of silicon ICs demonstrated both a unique substrate profiling and novel inspection technology for circuit navigation and characterisation. In addition, the application of OCT to the investigation of artwork samples and contemporary banknotes is demonstrated for the purposes of art conservation and counterfeit prevention

Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells

Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells. It forms a complex with both focal adhesion kinase (FAK, also known as PTK2) and Src in SCC cells derived from skin carcinomas induced by administration of the chemical DMBA followed by TPA (the DMBA/TPA model). Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with Src and drives trafficking of Src to autophagic structures that SCC cells use to cope with high levels of active Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, thereby meaning active Src becomes tethered at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration

FAK goes nuclear to control anti-tumor immunity – a new target in cancer immuno-therapy

Evading the antitumor immune response is important for the survival and progression of cancer. Recently, we identified an unexpected role for nuclear Focal Adhesion Kinase (FAK) activity in the control of tumor Treg levels and immune evasion by regulating chemokine and cytokine transcription in cancer cells. We proposed a potentially new purpose for FAK kinase inhibitors, which can cause immune-mediated tumor regression

Solid immersion lens applications for nanophotonic devices

Solid immersion lens (SIL) microscopy combines the advantages of conventional microscopy with those of near-field techniques, and is being increasingly adopted across a diverse range of technologies and applications. A comprehensive overview of the state-of-the-art in this rapidly expanding subject is therefore increasingly relevant. Important benefits are enabled by SIL-focusing, including an improved lateral and axial spatial profiling resolution when a SIL is used in laser-scanning microscopy or excitation, and an improved collection efficiency when a SIL is used in a light-collection mode, for example in fluorescence micro-spectroscopy. These advantages arise from the increase in numerical aperture (NA) that is provided by a SIL. Other SIL-enhanced improvements, for example spherical-aberration-free sub-surface imaging, are a fundamental consequence of the aplanatic imaging condition that results from the spherical geometry of the SIL. Beginning with an introduction to the theory of SIL imaging, the unique properties of SILs are exposed to provide advantages in applications involving the interrogation of photonic and electronic nanostructures. Such applications range from the sub-surface examination of the complex three-dimensional microstructures fabricated in silicon integrated circuits, to quantum photoluminescence and transmission measurements in semiconductor quantum dot nanostructures

Src/FAK-mediated regulation of E-cadherin as a mechanism for controlling collective cell movement Insights from in vivo imaging

Recent advances in confocal and multi-photon microscopy, together with fluorescent probe development, have enabled cancer biology studies to go beyond the culture dish and interrogate cancer-associated processes in the complex in vivo environment. Regulation of the tumor suppressor protein E-cadherin plays an important role in cancer development and progression, and may contribute to the decision between ‘single cell’ and ‘collective invasion’ in vivo. Mounting evidence from in vitro and in vivo experiments places the two nonreceptor protein tyrosine kinases Src and Focal Adhesion Kinase at the heart of E-cadherin regulation and the crosstalk between integrins and cadherins. Here we discuss recent insights, attained using high-resolution fluorescent in vivo imaging, into the regulation of E-cadherin and collective invasion. We focus on the regulatory crosstalk between the Src/FAK signaling axis and E-cadherin in vivo

Surface imaging of metallic material fractures using optical coherence tomography

We demonstrate the capability of optical coherence tomography (OCT) to perform topography of metallic surfaces after being subjected to ductile or brittle fracturing. Two steel samples, OL 37 and OL 52, and an antifriction Sn-Sb-Cu alloy were analyzed. Using an in-house-built swept source OCT system, height profiles were generated for the surfaces of the two samples. Based on such profiles, it can be concluded that the first two samples were subjected to ductile fracture, while the third one was subjected to brittle fracture. The OCT potential for assessing the surface state of materials after fracture was evaluated by comparing OCT images with images generated using an established method for such investigations, scanning electron microscopy (SEM). Analysis of cause of fracture is essential in response to damage of machinery parts during various accidents. Currently the analysis is performed using SEM, on samples removed from the metallic parts, while OCT would allow in situ imaging using mobile units. To the best of our knowledge, this is the first time that the OCT capability to replace SEM has been demonstrated. SEM is a more costly and time-consuming method to use in the investigation of surfaces of microstructures of metallic materials. © 2014 Optical Society of America

FAK and talin: Who is taking whom to the integrin engagement party?

In this issue, Lawson et al. provide new insight into the relationship between FAK and talin during assembly of integrin adhesions on fibronectin. They show that FAK is upstream of talin, and that talin is not required for FAK recruitment or for integrin activation at nascent adhesions. However, FAK-talin binding is required for adhesion turnover and cell motility. The findings question the view that talin is always upstream of focal adhesion protein recruitment to clustered integrin sites

Imaging Drug Uptake by Bioorthogonal Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy in tandem with bioorthogonal Raman labelling strategies is set to revolutionise the direct visualisation of intracellular drug uptake. Rational evaluation of a series of Raman-active labels has allowed the identification of highly active labels which have minimal perturbation on the biological efficacy of the parent drug. Drug uptake has been correlated with markers of cellular composition and cell cycle status, and mapped across intracellular structures using dual-colour and multi-modal imaging. The minimal phototoxicity and low photobleaching associated with SRS microscopy has enabled real-time imaging in live cells. These studies demonstrate the potential for SRS microscopy in the drug development process