The objective of this thesis is to present the fabrication of a multiphoton microscope and the underlying theory responsible for its proper functioning. A basic introduction to nonlinear optics will give the necessary knowledge to the reader to understand the optical effects involved. Femtosecond laser pulses will be presented and characterized. Each part of the microscope, their integration and the design of the microscope will be discussed. The basic concepts of laser scanning microscopy are also required to explain the design of the scanning optics. Fast scanning problems and their solutions are also briefly viewed. As a working proof, the first images taken with the microscope will be presented. Fluorescent beads, rat tail tendon, gold nanoparticles and pollen grain images using various nonlinear effects will be shown and discussed.

The main focus of this thesis is the characterization of defects and microstructure in high-energy ball milled magnesium hydride powder and magnesium-based multilayered composites. Enhancement in kinetics of hydrogen cycling in magnesium can be achieved by applying severe plastic deformation. A literature survey reveals that, due to extreme instability of -MgH2 in transmission electron microscope (TEM), the physical parameters that researchers have studied are limited to particle size and grain size. By utilizing a cryogenic TEM sample holder, we extended the stability time of the hydride phase during TEM characterization. Milling for only 30 minutes resulted in a significant enhancement in desorption kinetics. A subsequent annealing cycle under pressurized hydrogen reverted the kinetics to its initial sluggish state. Cryo-TEM analysis of the milled hydride revealed that mechanical milling induces deformation twinning in the hydride microstructure. Milling did not alter the thermodynamics of desorption. Twins can enhance the kinetics by acting as preferential locations for the heterogeneous nucleation of metallic magnesium. We also looked at the phase transformation characteristics of desorption in MgH2. By using energy-filtered TEM, we investigated the morphology of the phases in a partially desorbed state. Our observations prove that desorption phase transformation in MgH2 is of nucleation and growth type, with a substantial energy barrier for nucleation. This is contrary to the generally assumed core-shell structure in most of the simulation models for this system. We also tested the hydrogen storage cycling behavior of bulk centimeter-scale Mg-Ti and Mg-SS multilayer composites synthesized by accumulative roll-bonding. Addition of either phase (Ti or SS) allows the reversible hydrogen sorption at 350C, whereas identically roll-bonded pure magnesium cannot be absorbed. In the composites the first cycle of absorption (also called activation) kinetics improve with increased number of fold and roll (FR) operations. With increasing FR operations the distribution of the Ti phase is progressively refined, and the shape of the absorption curve no longer remains sigmoidal. Up to a point, increasing the loading amount of the second phase also accelerates the kinetics. Microscopy analysis performed on 1-2 wt.% hydrogen absorbed composites demonstrates that MgH2 formed exclusively on various heterogeneous nucleation sites. During activation, MgH2 nucleation occurred at the Mg-hard phase interfaces. On the subsequent absorption cycles, heterogeneous nucleation primarily occurred in the vicinity of internal free surfaces such as cracks. / Materials Engineering

Human bone is considered one of the most direct and insightful sources of information on peoples of the past. As a result, curation protocols have been developed to ensure that the integrity of human skeletal collections is maintained. Although collections are generally considered safe when these protocols are followed, the results of this investigation show that the Tell Leilan skeletal collection from Syria (circa 2900 – 1900 BCE) was contaminated by microbial growth (also known as biodeterioration) during curation. This biodeterioration was evaluated by light microscopy (LM), by the application of a histological preservation index (HPI), and by scanning electron microscopy (SEM). All samples (n=192) were found to be biodeteriorated by LM and the HPI. SEM confirmed that the Tell Leilan skeletal material had been contaminated by a complex microbial aggregate known as a biofilm. Amycolatopsis sp. and Penicillium chrysogenum, along with species of Aspergillus, Chaetomium, and Cladosporium were isolated and cultured from several contaminated bones and were identified based on morphology and DNA sequences. The results of this research suggest that we must focus on new techniques to examine bone as well as on new conservation protocols designed to limit the growth of biofilms in human skeletal collections in the future.

We have designed and engineered bi-catalyst magnesium hydride composites with superior sorption performance to that of ball milled magnesium hydride catalyzed with the individual baseline catalysts. We have examined the effect of single-walled carbon nanotube (SWCNT)-metallic nanoparticle additions on the hydrogen desorption behavior of MgH2 after high-energy co-milling. We showed the synergy between SWCNT's and metallic nanoparticles in catalyzing the sorption of magnesium hydride. The optimum microstructure for sorption, obtained after 1 h of co-milling, consists of highly defective SWCNTs in intimate contact with metallic nanoparticles and with the hydride. This microstructure is optimum, presumably because of the dense and uniform coverage of the defective SWCNTs on the MgH2 surface. Cryo-stage transmission electron microscopy (TEM) analysis of the hydride powders revealed that they are nanocrystalline and in some cases multiply twinned. Since defects are an integral component of hydride-to-metal phase transformations, such analysis sheds new insight regarding the fundamental microstructural origins of the sorption enhancement due to mechanical milling. The nanocomposite shows markedly improved cycling as well. Activation energy analysis demonstrates that any catalytic effect due to the metallic nanoparticles is lost during cycling. Improved cycling performance is instead achieved as a result of the carbon allotropes preventing MgH2 particle agglomeration and sintering. The nanocomposite received over 100 sorption cycles with fairly minor kinetic degradation. We investigated the catalytic effect of Fe + Ti bi-metallic catalyst on the desorption kinetics of magnesium hydride. Sub-micron dimensions for MgH2 particles and excellent nanoscale catalyst dispersion was achieved by high-energy milling. The composites containing Fe shows DSC desorption temperature of 170 °C lower than as-received MgH2 powder, which makes it suitable to be cycled at relatively low temperature of 250 °C. The low cycling temperature also prevents the formation of Mg2FeH6. The ternary Mg-Fe-Ti composite shows best performance when compared to baseline ball milled magnesium hydride with only one catalytic addition. With a very high BET surface area it also shows much less degradation during cycling. The synergy between Fe and Ti is demonstrated through use of TEM and by carefully measuring the activation energies of the baseline and the ternary composites. / Materials Engineering

Bone response is one of the key determining factors in the overall success of biomaterials intended for bone regeneration and osseointegration. Understanding the formation of bone at an implant surface may lead to the improved design of biomaterials for the future. However, due to the inhomogeneity of bone tissue at an interface, two-dimensional images often lack detail on the interfacial complexity. Furthermore, the increasing use of nanotechnology in the design and production of biomaterials demands characterization techniques on a similar nano length scale. While current analysis methods, such as X-ray tomography, transmission electron microscopy, focused ion beam microscopy and scanning electron microscopy, provide a basis for analysing biomaterials and biointerfaces, they are incapable of doing so with both nanometre resolution and three-dimensional clarity. In contrast, electron tomography may be used to characterize the three-dimensional structure of biomaterials and their interfaces to bone with nanometre resolution. In this work, hydroxyapatite scaffolds, and laser-modified titanium and Ti6Al4V implants were studied in contact with human or rabbit bone. Z-contrast electron tomography revealed that the orientation of collagen in bone apposing hydroxyapatite, titanium and Ti6Al4V implants is consistently parallel to the implant surface, where the bioactive layer that precipitates on HA is oriented perpendicular to the implant surface. With this method, complete three-dimensional nanoscale osseointegration of titanium-based implants was also established. The extension of this technique from interfacial analyses to the design of biomaterials provided an understanding of the pore structure of mesoporous titania. In further investigations, the open three-dimensional pore network, as revealed by electron tomography, showed promise as a coating that improves implant osseointegration and enables site-specific drug-delivery from an implant surface. In summary, it was demonstrated that two-dimensional characterization techniques were insufficient for the investigation of nanostructured biomaterials, as well as their interfaces to bone. Visualizing biointerfaces and biomaterials with nanometre precision in three-dimensions can expose new fundamental information on materials properties and bone response, enabling better design of biomaterials for the future.

Coronary artery disease is a leading cause of morbidity and mortality in developed countries. Percutaneous transluminal coronary angioplasty (PTCA) is an important treatment option when intervention is required; namely for patients with relatively severe occlusions. However, adverse events including recurrence of angina pectoris and restenosis of the treated artery limit patient prognosis, with subsequent re-vascularisation often necessary. Platelet activation accompanies PTCA, with platelet adhesion and aggregation involved in thrombus formation during restenosis. During platelet activation, highly coagulant platelet-derived microparticles (PMPs) are formed, and it is likely that these PMPs will also be produced during PTCA. While platelet aggregation inhibitors used during PTCA limit platelet aggregation and decrease the incidence of restenosis, they do not prevent PMPs being formed. PMPs are capable of adhesion and aggregation, and adhere to PTCA treated arteries in an animal model. Therefore, in order to understand the phenomenon of restenosis and its improved limitation, it is necessary to investigate PMP formation during PTCA. The field of PMP study is in its infancy, with conflicting results from the substantial inequities in methods of PMP measurement, which may be exacerbated by PMP heterogeneity. The current literature on this topic is reviewed in Chapter 2, where the PMP surface and possible functions are considered, and the PMP size and morphology examined. To conclude, the relationship between PMPs and PTCA is explored, with a focus on the potential role of PMPs in restenosis. The knowledge deficiencies in this field are highlighted at the conclusion of this chapter. Very little is known regarding the production of PMPs with PTCA. The level of PMPs during PTCA was monitored in paired arterial blood samples obtained from seventy-five patients undergoing the procedure (Chapter 3). A significant increase in PMPs from baseline to completion of PTCA was clearly demonstrated for the first time. This indicated that procoagulant PMPs are produced during PTCA and may contribute to subsequent restenosis. Furthermore, administration of the platelet aggregation inhibitor abciximab to a group of thirty-eight high risk patients limited PMP formation; given that abciximab patients required more rigorous PTCA, the protective benefit of this medication for PMP production is underlined. Although few patients in this study experienced restenosis, it is interesting to note that of those treated with abciximab, all patients suffering subsequent restenosis were revascularised using PTCA. This demonstrates that their occlusions were comparatively mild as the need for coronary artery bypass grafting was avoided, and suggests that minimisation of PMP levels may assist in limiting the progression of severe restenosis. The increased peripheral level of PMPs predicated investigation of the coronary circulation to determine local events. Although the level of PMPs increased significantly within the coronary arteries of PTCA patients, there was no corresponding increase in the coronary sinus (Chapter 4). This important finding indicated that significant levels of PMPs remained within the coronary circulation, where their ability to adhere, aggregate and accelerate haemostasis may allow them to contribute directly to restenosis. During the time when increased levels of PMPs were being formed, there was no evidence of platelet lysis, which refuted the hypothesis that PMPs are merely membrane fragments of lysed platelets. A wide variation in reported PMP sizes has contributed to the hypothesis that PMPs are heterogeneous. As morphological information can assist in understanding physiology, the final study was designed to investigate platelet morphology from PTCA patients (Chapter 5). Most platelets were activated prior to and following PTCA, with a slight decrease in body size occurring due to PTCA, presumably due to loss of cytoplasm in PMPs being shed as reported in the previous chapter. Importantly, platelet distal pseudopod buds were observed, and these did not alter significantly with PTCA. These buds were probably unformed PMPs, although the exact mechanism of PMP formation remains undetermined. The platelet pseudopods were longer and significantly thinner distally with PTCA, which may be due to movement of cytoplasm into these terminal swellings. In addition, buds or swellings directly on the platelet body were smaller following PTCA, and it is likely these may also be PMPs prior to detachment from the parent platelet. This work has contributed substantially to knowledge of PMPs produced during PTCA. The level of PMPs increased significantly in peripheral arterial samples, with the platelet aggregation inhibitor abciximab preventing this occurrence. This may indicate that functional aggregation receptors are an essential requirement for PMP formation under these conditions. However, it is possible for PMPs to be formed when aggregation is inhibited, and therefore the molecular mechanisms of PMP formation remain unconfirmed. The examination of PMPs from the coronary circulation provided valuable data indicating that PMPs are produced during PTCA but remain within the coronary circulation. As PMPs are capable of adhesion and aggregation, this strongly suggests that PMPs within the coronary circulation would contribute directly to pathogenesis of restenosis, although further investigation on PMPs with PTCA is necessary to confirm this association. The examination of platelet morphology during PTCA indicated that platelets possessed terminal pseudopod swellings, and cell surface swellings. Importantly, the terminal swellings, which are likely to be unformed PMPs, were observed for the first time during PTCA.

The influence of steel metallurgy on rock bolt SCC was studied using a series of commercial carbon and low-alloyed steels. The chemical composition, their mechanical properties and the microstructures of these steels varied considerably in order to gather information for the discussion of the metallurgical influences under Hydrogen Embrittlement (HE) and Stress Corrosion Cracking (SCC) conditions. In order to understand the metallurgical influences on Rock Bolt SCC, an evaluation was carried out to fifteen commercial steels. The experiments reproduced the Stress Corrosion Cracking condition at which commercial rock bolts had failed in Australians mines. Due to the selected materials, stress and electrolyte condition it is expected that Hydrogen Embrittlement (HE) will affect the steel failure. The approach was to use the Linearly Increasing Stress Test (LIST) and exposing the sample to a dilute pH 2.1-sulphate solution, in accordance with prior studies. Stress Corrosion Cracking was evaluated by analysing the decrease in tensile strength, loss of ductility and fractography observed using Scanning Electron Microscopy (SEM). The initial series of test to the fifteen steels were performed at the free corrosion potential (f.c.p.) vs. Ag/AgCl. From this initial test only five steels (AISI 1008, AISI 4140, AISI 4145H, pipeline X-65 and X-70) did not show Stress Corrosion Cracking features. These five steel were tested in accordance with the Linearly Increased Stress Test (LIST) in the dilute pH 2.1 sulphate solution at different electronegative applied potential to minimum value of -1500mV. The experimental procedure tried to reproduce the Stress Corrosion Cracking condition to identify the most aggressive condition the steel is able to support before failing due to Stress Corrosion Cracking to then compare the theory of SCC and HE in low carbon and low alloy steel with the obtained experimental results. The investigation compared the well-known theory of SCC and HE in low carbon and low alloy steel with the obtained experimental results. Surprisingly, the experimental result did not always agree with the theory.

Acacia holosericea A. Cunn. Ex G. Don (Velvet Leaf Wattle, Family Mimosaceae) is indigenous to Australia and holds promise as a novel cut foliage crop due to its silvery green silky phyllodes. Insufficient water uptake, possibly due to low stem hydraulic conductivity (Kh), is potentially responsible for early wilting and desiccation of phyllodes and limiting vase life. This study aimed to characterize the anatomy of stem xylem conduits and determine cation (K+ and Ca2+) mediated stem Kh. Differential localization of Ca2+ in xylem vessels and the effects of KCl and CaCl2 salts as cation contributors in vase solutions were also evaluated for their effects on cut foliage longevity. Anatomical characteristics of stem xylem conduits were studied using light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Tracheids and vessels with simple perforation plates were the principal water conducting cells. SEM and TEM revealed bordered vestured intervessel pits in xylem conduits. Vestures were branched and coralloid-like structures. Xylem conduit lengths were assessed by ink perfusion. The majority of conduits (89%) were short (1 to 5 cm), and only ~ 2% were long (15 to 16 cm). Mean conduit diameter was 77 ± 0.9 µm, with 29% of conduits in the range 70 to 80 µm. Effects of S-carvone (0, 0.318, and 0.636 mM), a monoterpene inhibitor of wound-healing enzyme inhibitor found in caraway (Carum carvi) and dill (Anethum graveoleus) seeds, on several native Australian, non-proteaceous cut flower and foliage species including A. holosericea (Mimosaceae), Baeckea frutescens (Myrtaceae), Chamelaucium uncinatum cv. ‘Mullering Brook’ (Myrtaceae), and the non-native Chrysanthemum sp. cv. ‘Dark Splendid Reagan’ (Asteraceae) were examined. As comparator treatments regular recutting of stem ends and use of standard tap water (STW: 0.7 mM CaCl2, 1.5 mM NaHCO3, and 0.05 mM CuSO4 in vases) were tested. S-Carvone treatments significantly (P≤0.05) extended the vase life of B. frutescens and C. uncinatum, constituting the first report of positive S-carvone effects on the vase life of Myrtaceous species. S-Carvone at 0.318 and 0.636 mM did not have antibacterial effects against Bacillus cereus (the main vase solution microbe) either in vitro or in the vase solution. Regular recutting of stem ends consistently improved all vase life parameters [viz. relative fresh weight (RFW), solution uptake, and vase life] in the three native species examined. STW had a positive effect on RFW and solution uptake only for A. holosericea cut foliage. Effects of di- and monovalent cations (Ca2+ as CaCl2 and K+ as KCl) on stem Kh of cut stem segments were studied. Abundance of Ca2+ on pit membranes versus xylem lumen wall surfaces was investigated using a novel low vacuum (LV) SEM plus energy dispersive X-ray (EDX) microanalysis technique. Both salts (0.1, 1, 10, and 100 mM KCl or CaCl2) did not significantly increase stem Kh compared to the corresponding deionised (DI) water controls (experiment 1). Highest increase in Kh was with KCl and CaCl2 at 10 and 1 mM, respectively. Increases in Kh with 100 mM KCl and CaCl2 were significant over DI water (experiment 2) for long (10 and 20 cm) and short (2 and 5 cm) stem segments, respectively. Increases in Kh of 1.2- and 2.4-fold for 100 mM KCl over DI water were found with increasing stem length from 2 to 20 cm. Kh decreased as stem segment length increased from 5 to 20 cm. However, contrasting results were found with 100 mM CaCl2, where ΔKh was larger in shorter (2 cm) than longer (20 cm) segments. To prevent dislocation of ions and distortion damage to the specimens, stem pieces were first LV freeze-dried, and then carbon-coated, viewed under SEM, and analysed for elemental composition and distribution by EDX. However, the method could not identify specific calcium peaks in xylem vessels perhaps because background signals were too high, and tissue topography interfered with signal detection. Effects of KCl and CaCl2 on vase life were also tested. RFW, solution uptake, and vase life were higher with 10 mM KCl and CaCl2 in the vase solution than with 0, 1, and 100 mM. STW had a significant (P≤0.05) positive effect on RFW and solution uptake rate when tested against deionised water, 10 mM KCl and 10 mM CaCl2. Different combinations of 0.05 mM CuSO4, 10 mM CaCl2, and 10 mM KCl were also tested as vase solutions. A significant positive effect on RFW and vase life was obtained with CuSO4 alone, CaCl2 alone, and CuSO4 plus KCl. Only CuSO4 and CaCl2 gave a significant positive effect on solution uptake rate. None of the eight treatments tested showed a consistent effect on stomatal conductance or stem Kh. Overall, the research revealed that tracheids and vessels with simple perforation plates and bordered vestured intervessel pits are the principal water conducting cells in A. holosericea. LV-SEM-EDX technique was unable to assess the spatial distribution of Ca2+ on xylem vessels, but this was the first attempt to apply this technique. Also, this is the first report on the extension of vase life of B. frutescens and C. uncinatum (Myrtaceous species) using S-carvone. In vase solutions, 0.05 mM CuSO4, 10 mM KCl plus 0.05 mM CuSO4 and 10 mM CaCl2 should have positive influences on the water balance of A. holosericea cut foliage stems.

Developmental studies on elasmobranchs are challenging due to the difficulties in obtaining sufficient numbers of animals of different age cohorts. The brown banded bamboo shark, Chiloscyllium punctatum is a good model in this regard as it is abundant and readily available in the wild, is quick to mature, is frequently housed and bred in captivity and is a relatively small species of shark. Whilst there are important factors that must be considered when comparing the retinal development of animals raised in captivity to those caught from the wild, the use of C. punctatum represents an outstanding opportunity to study the development of the elasmobranch visual system from pre-hatching embryonic to adult life stages. In this study, the developing eye and retina of C. punctatum were studied using light and electron microscopy, electroretinography (ERG) and microspectrophotometry (MSP). To provide a source of early-stage animals, and to investigate the effects of environmental factors (such as temperature) on physical development, a captive breeding program was established at the University of Queensland. Sharks sourced from this facility were supplemented with animals bred at UnderWater World, Sea World and caught from the wild. Monitoring the fecundity, embryonic development, growth and viability of captive C. punctatum showed that females lay an average of 115.3 eggs, 38 of which were viable and 21.4% of which hatched. Embryos have an average gestation of 153 days post deposition (dpd; temp: 21 - 25º C) and embryonic growth is most rapid from 99 dpd until hatching. The eye of C. punctatum develops early in embryogenesis, with visible optic vesicles bulging at 27 dpd. Recent advances in fixation and processing techniques for transmission electron microscopy (TEM) have yielded improved levels of ultrastructural detail in a variety of tissue types. Consequently, in addition to conventional chemical fixation (CF) methods, the retina of C. punctatum was also processed using microwave chemical fixation (MCF) and high pressure freezing (HPF), and the resulting ultrastructure compared. Both MCF and HPF produced superior retinal ultrastructure compared to conventional CF, evidenced by higher resolution of ultrastructural detail and fewer artefacts. MCF provided the best, consistent ultrastrucutral results. By examining the time-course of retinal cell differentiation, it was found that ganglion and Müller cells are the first to differentiate, at approximately 81 dpd. The interneurons differentiate next, beginning with the amacrine cells (81 dpd), followed by the bipolar cells (101 dpd) and horizontal cells (124 dpd). The adult retina is duplex and rod and cone photoreceptors are differentiated and synaptic connections are formed by 124 dpd. Topographic analysis of retinal neuron sub-types reveals that C. punctatum undergoes rapid changes in ganglion cell distribution during embryogenesis. High levels of apoptosis, especially around the retinal periphery, result in relatively higher cell densities in the central retina, which progressively extend nasally and temporally to form a meridional band. C. punctatum develops a horizontal streak and shows only minor changes in topography during growth. Only basal levels of apoptosis are seen post-hatching. In the adult shark, the total ganglion cell number reaches 547,881. The mean and highest retinal ganglion cell densities reach a peak around hatching (3,228 cells mm-2 and 4,983 cells mm-2, respectively). Using measurements of lens focal length and ganglion cell density, the calculated maximum spatial resolving power (assuming a hexagonal mosaic) increases from 1.47 cycles degree-1 during embryogenesis to 4.29 cycles degree-1 in adults. The addition of a high ganglion cell density area within the visual streak and an increasing spatial resolving power over post-hatching development suggest an increased prey targeting and capture ability for this species. Using ERG, it is shown that C. punctatum becomes responsive to light at 127 dpd and light sensitivity peaks around the time of hatching, with a slight decrease post-hatching. C. punctatum maintains a flicker fusion frequency (FFF; an indicator for temporal resolution) at 7 - 22 Hz through juvenile stages), which is relatively low compared to other marine predators. ERG results suggest that this species is adapted to low light vision with low temporal resolution. The early differentiation, development and functionality of the visual system in C. punctatum allows for a period of synaptic maturation and potentially the ability of embryonic predator avoidance. The retina of C. punctatum contains a rod visual pigment with a wavelength of maximum absorbance (λmax) at 500 nm and cone visual pigment with a λmax at 532 nm; the max values of these pigments do not change during development. Rod and cone outer segments differentiate at 113 days post deposition (dpd), lengthen during embryogenesis and accumulate pigment throughout life. Although the photoreceptors develop and differentiate well in advance of hatching, there is considerable variation in outer segment length and pigment density during embryogenesis, which suggests that these cells are developing up until hatching. C. punctatum does not appear to have the potential for colour vision based on the lack of two cone photoreceptor types each containing a visual pigment maximally sensitive to different parts of the visual spectrum, but appears specialised for dim-light contrast vision.

The deformation mechanisms operating in diamond-like carbon (DLC) coatings on (100) and (111) Si, has been investigated. The effect of coating thickness, indenter geometry, substrate orientation and deposition technique on the deformation of DLC coatings and the underlying substrate was studied by undertaking nanoindentation followed by subsurface microstructural characterization. Uncoated (111) Si was also investigated for comparison. The observed microstructural features were correlated to the indentation response of the coatings and compared with simulation studies, as well as observations on uncoated Si. In uncoated (111) Si, phase transformation was found to be responsible for the discontinuities in the load-displacement curves, similar to (100) Si. However, slip was activated on {311} planes instead of on {111} planes. Moreover, the density of defects was also significantly lower and their distribution asymmetric. The coatings were adherent, uniformly thick and completely amorphous. The load-displacement curves displayed several pop-ins and a pop-out, the indentation loads for the first pop-in and the pop-out depending primarily on the thickness of the coating. The coatings exhibited localized compressive deformation in the direction of loading without any through-thickness cracks. The extent of this localized deformation increased with indentation load. Hardness and thickness of the coatings and the geometry of the indenter influenced the magnitude of compressive strains. Harder and thinner coatings and a blunt indenter exhibited the minimum degree of deformation. Densification by rearrangement of molecules has been suggested as the mechanism responsible for plastic compression. At indentation loads corresponding to the first pop-in, (100) and (111) silicon substrates initially deformed by <111> and <311> slip respectively. Higher indentation loads caused phase transformation. Therefore, unlike in uncoated Si, dislocation nucleation in the Si substrate has been proposed as the mode responsible for the first pop-in. Subsequent pop-ins were attributed to further deformation by slip and twinning, phase transformation and extensive cracking (median and secondary cracks) of the substrate. The pop-out, however, was ascribed to phase transformation. Extensive deformation in the substrate, parallel to the interface, is attributed to the wider distribution of the stress brought about by the DLC coating. Good correlation was obtained between the nanoindentation response, microstructural features and simulation studies.