Nanometrology

Methods and protocols are described when using fluorescence metrology to determine the average nanoparticle (np) size in colloids in the range of 1–10 nm. The technique is based on determining the rotational correlation time of the np from the decay of fluorescence anisotropy of a dye that is electrostatically or covalently attached to the np as it undergoes Brownian rotation. The np size is then calculated from the Stokes–Einstein equation. The exemplar of silica nps is presented, but the approach can also be applied to other types of nps

Mapping the formation of eumelanin using coupled measurements

Melanin plays a crucial role as a pigment all through the animal kingdom. Being a macromolecule just on the divide between an ordered crystalline or a purely amorphous form melanin has proven a challenge to structure-function analysis. Melanin assembles from small molecules much like a jigsaw and much like in a jigsaw the fine detail quickly vanishes in the overall picture. With Melanin being first and foremost a photo-active molecule we focus on spectral properties for the characterization of its structure using linked measurements of excitation and emission to identify ‘areas of interest’ in the Excitation-Emission Matrix (EEM). We then probe for characteristic fluorescence lifetimes in the identified areas to track melanin building blocks through the formation pathway

Tracking the formation of eumelanin from L-Dopa using coupled measurements

Melanin plays a crucial role as a pigment all through the animal kingdom. Being a macromolecule just on the divide between an ordered crystalline or a purely amorphous form melanin has proven a challenge to structure-function analysis. Melanin assembles from small molecules much like a jigsaw and much like in a jigsaw the fine detail quickly vanishes in the overall picture. With melanin being first and foremost a photo-active molecule we focus on spectral properties for the characterisation of its structure. We use absorption measurements to illustrate the complex nature of the formation process. To gain a better hold on the formation pathway we use coupled measurements of excitation and emission to identify 'areas of interest' in the excitation-emission matrix (EEM). We then probe one area for characteristic fluorescence lifetimes to track one melanin building block through the formation process. Comparison of the EEMs of L-Dopa derived melanin with natural Sepia melanin shows characteristic differences. We show how the presence of copper ions creates a melanin closer to its natural form

Friction stir processing of aluminium-silicon alloys

Friction Stir Processing (FSP) has the potential for locally enhancing the properties of Al-Si alloy castings, for demanding applications within the automotive industry. In this thesis, the effect of FSP has been examined on three different cast Al-Si alloys:i) A Hypoeutectic Al-8.9wt%Si Alloyii) A Hypereutectic Al-12.1wt%Si Alloyiii) A Hypereutectic Al-12.1wt%Si-2.4wt%Ni AlloyThe influence of different processing parameters has been investigated at a fundamental level. Image analysis of particle size distributions and growth method of tessellation were used to quantify the level of particle refinement and the homogeneity of the second phase spatial distribution. Stop-action experiments were also carried out, to allow the microstructural changes around the tool during FSP to be studied. Two computer models have been explored, in order to predict the temperature distribution and the material flow behaviour. Furthermore, the stability of the microstructure of the friction stir processed material was studied after being heat treated at elevated temperatures. The changes in particle size and grain structure were examined, hardness measurements were taken across the PZ, and tensile testing were carried out at room and elevated temperatures.After FSP, the microstructure of the cast Al-Si alloys was greatly refined. However, differences in microstructure have been observed throughout the PZ, which tended to be better refined and distributed on the advancing side, than the retreating side of the PZ. Changing the processing parameters also influenced the size and spatial distribution of the second phase particles. By studying the changes in microstructure around the tool from the stop-action experiments, and comparing the results to the thermal distribution and material flow behaviour predicted by the computer models, it has been shown that the flow stress, pitch, and temperature of processing, all needed to be considered, when determining the effects that FSP have on the microstructure. FSP caused very little changes to the hardness of the material, while tensile properties were greatly improved, due to the elimination of porosity and refinement of large flawed particles. In terms of the stability of the microstructure after FSP, particle coarsening and abnormal grain growth has been observed during high temperatures heat treatment. Furthermore, the Al2Cu phase was found to dissolve into solid solution at elevated temperatures, so GPZs and solute clustering can then develop within the alloy during natural ageing.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Influence of ions and pH on formation of solid and liquid-like melanin

Melanin is a natural pigment with broadband absorption and effective ability to dissipate the energy absorbed. The macromolecular structure of melanin shows a delicate balance between short-range ordered and disordered structures without being a random aggregate. The presence of ions or the variation in pH or ionic strength can alter the self-assembly process which subsequently changes the structure of melanin. To understand these relationships, this study investigates the influence of ions and pH in melanin formation. The types of ions present and pH have a profound influence on the formation and structure of melanin particles, while only minor changes are observed in the absorption and excitation-emission analysis. In some conditions, the formation of discernible particles with significant refractive index contrast is avoided while retaining the spectroscopic characteristics of melanin, leading to liquid-like melanin. These findings identify potential pathways which can be used to manipulate the melanin macromolecular structure while providing the desired spectral properties to enable novel bio-engineering applications.

Nanoparticle metrology of silica colloids and super-resolution studies using the ADOTA fluorophore

We describe how a new fluorescent dye, methyl ADOTA (N-methyl-azadioxatriangulenium tetrafluoroborate), is an improvement on dyes reported previously for measuring silica nanoparticle size in sols using the decay of fluorescence anisotropy. Me(thyl)-ADOTA possesses the unusual combination of having a red emission and a long fluorescence lifetime of ~ 20 ns, leaving it better-placed to reveal particle sizes at the upper end of the 1-10 nm measurement range. For stable LUDOX colloids, Me-ADOTA is shown to offer higher measurement precision in ≤ 1/30th of the measurement time required for dyes previously used. In measurement times of only ~ 20 mins nanoparticle radii for LUDOX SM-AS, AM and AS-40 of 4.6 ± 0.3 nm, 5.9 ± 0.2 nm and 11.1 ± 1.1 nm, are in good agreement with two of the manufacturer’s values of 3.5 nm, 6 nm and 11 nm respectively. Unlike the Si-ADOTA (N-(4-(triethoxysilylethyl)urea-phenyl-) ADOTA tetrafluoroborate) derivative containing a reactive trimetoxysilane group, Me-ADOTA is shown to not induce aggregation of colloidal silica. Measurements on nanoparticles growing in an acidic silica hydrogel at pH 0.94, prior to the gel time of ~ 50 hr, reveals an average nanoparticle size up to ~ 6.3 nm, significantly larger than the 4.5 nm reported previously. The difference is most certainly due to the longer fluorescence lifetime of Me-ADOTA (~ 20 ns) revealing the presence of larger particles. Studies of growing silica clusters in an alcogel of tetraethyl orthosilicate (TEOS) were able to resolve a monotonically increasing average radius of 1.42 ± 0.10 nm to 1.81 ± 0.14 nm over a period of 48 hr. We have also assessed a carboxylic acid derivative of ADOTA (N-(3-carboxypropylene)-ADOTA tetrafluoroborate - Acid-ADOTA) using dSTORM super-resolution microscopy. Although demonstrating high photochemical stability and blinking, its lower brightness and relative propensity to aggregate limits Acid-ADOTA’s use for dSTORM

Identification of specificity determining residues in peptide recognition domains using an information theoretic approach applied to large-scale binding maps

<p>Abstract</p> <p>Background</p> <p>Peptide Recognition Domains (PRDs) are commonly found in signaling proteins. They mediate protein-protein interactions by recognizing and binding short motifs in their ligands. Although a great deal is known about PRDs and their interactions, prediction of PRD specificities remains largely an unsolved problem.</p> <p>Results</p> <p>We present a novel approach to identifying these Specificity Determining Residues (SDRs). Our algorithm generalizes earlier information theoretic approaches to coevolution analysis, to become applicable to this problem. It leverages the growing wealth of binding data between PRDs and large numbers of random peptides, and searches for PRD residues that exhibit strong evolutionary covariation with some positions of the statistical profiles of bound peptides. The calculations involve only information from sequences, and thus can be applied to PRDs without crystal structures. We applied the approach to PDZ, SH3 and kinase domains, and evaluated the results using both residue proximity in co-crystal structures and verified binding specificity maps from mutagenesis studies.</p> <p>Discussion</p> <p>Our predictions were found to be strongly correlated with the physical proximity of residues, demonstrating the ability of our approach to detect physical interactions of the binding partners. Some high-scoring pairs were further confirmed to affect binding specificity using previous experimental results. Combining the covariation results also allowed us to predict binding profiles with higher reliability than two other methods that do not explicitly take residue covariation into account.</p> <p>Conclusions</p> <p>The general applicability of our approach to the three different domain families demonstrated in this paper suggests its potential in predicting binding targets and assisting the exploration of binding mechanisms.</p

Molecular rheometry: direct determination of viscosity in L-o and L-d lipid phases via fluorescence lifetime imaging

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol–sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts