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• W3044805427 abstract "The work in this thesis was motivated by three main questions, namely: i) howfar can quantitative differential interference contrast (DIC) microscopy be expandedand improved to accurately measure the thickness of single lipid bilayers, ii) doGold nanoparticle-fluorophore conjugates provide a stable and reliable dual probefor correlative imaging studies, iii) can DIC offer a label-free quantitative methodto accurately measure the surface temperature of a Gold nanoparticle (AuNP). Toaddress the first question, a software has been developed which extracts opticalphase information from a specimen imaged with differential interference contrast. AWiener filtering approach is used to integrate the differential phase contrast image,to obtain an optical phase image of the specimen. The quality of Wiener filteredimages is improved through the use of an apodization process to produce a twodimensional window around the differential phase image similar to the Hann windowoften used in one dimensional Fast Fourier Transform algorithm. Additionally,an energy minimisation routine further improves the quality of the retrieved phaseimages. From results presented in this thesis, it has been shown that one can performthe energy minimisation algorithm on multiple images in parallel with variedparameters. Images reach a converged status after approximately 107 iterations.To perform 107 iterations on graphics processing units (GPUs), it takes approximately72 hours. To address the second question, following the acquisition of aseries of confocal fluorescence and four wave mixing (FWM) amplitude images frombioconjugated fluorescently labelled AuNPs in Naya Giannakopoulou’s thesis [1], across-correlation algorithm has been developed to quantify the Pearson’s coefficientfor lateral shifts between these images. To accommodate for any angular discrepanciesbetween these images, a further algorithm was developed in order to rotateimages relative to one another so that cross the images may be cross correlated.Pearson’s coefficients from cross correlated images showed a lack of colocalisationbetween FWM amplitude and fluorescence images of HeLa cells loaded with AuNPfluorophoreconstructs, generally resulting in values rP < 0.05 (apart from one instancewhere rP = 0.15) which confirms what was qualitatively reported in [1]. Adirect study of the correlation between extinction and epi-fluorescence images ofAuNP-fluorophore constructs prepared (washed) as per manufacturer specificationand deposited onto a glass surface was subsequently performed. The cross cor-– ix –relation of extinction and epi-fluorescence images from a 10nm AuNP-fluorophoreconstruct (10nmAuNP-SA(A-488)) gave Pearson’s coefficients rP < 0.06 for 1× and3× washes, clearly indicating that the supposedly attached fluorophores are not reliablereporters of the NP location. Conversely, 20nm diameter AuNPs covalentlybound to fluorescently labelled antibodies revealed a good degree of colocalisationwith the fluorophore (rP = 0.476) after 3× washes. To answer the third question,a AuNP heating set-up was developed, using the qDIC imaging method outlined inthis thesis. Briefly, a lipid bilayer was overlaid above a AuNP bound to a coverslip,and was photothermally heated at the localised surface plasmon resonance (LSPR).The heat which dissipates from the AuNP induces a phase transition in the lipidbilayer in its vicinity. By locating the phase boundary in the lipid bilayer, one canquantitatively measure the surface heating of the AuNP. In principle, the preciselocation of a phase boundary in a lipid bilayer is observable in qDIC. However, thedynamics observed through acquiring a qDIC time course did not resemble the behaviourpreviously observed in fluorescence measurements [2]. In particular, it ishypothesized that a blister effect occurred during resonant photothermal heating ofa 50nm AuNP at the LS" @default.
• W3044805427 created "2020-07-29" @default.
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• W3044805427 date "2019-09-30" @default.
• W3044805427 modified "2023-09-27" @default.
• W3044805427 title "Quantitative optical microscopy of bio-nanostructures" @default.
• W3044805427 hasPublicationYear "2019" @default.
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