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The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer.

Fluorescence microscopy has been widely used in characterizing the pathological states of tissues because intensity and spectra arise from fluorescence emission can reveal structural and biochemical information of biological samples and the fluorescence excited state lifetime has been verified to identify tissue pathology due to its sensitivity to the fluorophore microenvironment. In this study, we have demonstrated that early cervical cancer can be quantitatively diagnosed using intensity and lifetime derived from the stroma fluorescence in conjunction with extreme learning machine (ELM) classifier which can result in a concurrently high sensitivity of 99.1% and specificity of 99.6%. The results suggest that the proposed technique can be used to aid and supplement the traditional histopathological examination of cervical precancer.

Fluorescence Lifetime Imaging (FLIM) was used to study the histopathological conditions of cervical biopsy tissues. Measurements were conducted on more than 40 H&E stained cervical tissue sections. The characteristic decay lifetimes of the samples were extracted using an Expectation-Maximization and Bayesian Information Criterion algorithm. Diagnostic criterion based on the Extreme Learning Machine was developed to discriminate between normal and neoplastic samples. A high sensitivity and specificity of more than 80%were obtained. The proposed technique can be used to automate and supplement the traditional histopathological examination of cervical tissues.

Optical coherence tomography (OCT) is an optical imaging technique that allows the visualization of living tissues in vivo. However, the speckle noise affects their diagnostic accuracy by degrading the quality of OCT images. Wavelet based denoising methods have been successful in reducing the speckle noise in past but they require a manual selection of few parameters for optimum denoising. We propose a computerized method to select these parameters by using an evolutionary technique called differential evolution (DE). The selection process is purely objective and does not require any assumption of parameters as opposed to the existing wavelet based denoising methods. The performance improvement by using our method has been quantified in terms of signal to noise ratio (SNR) improvement and resolution preservation. An improvement of more than 17% in signal to noise ratio(SNR) was achieved while the resolution was restored after the application of our method.

Two optical coherence tomography (OCT) systems were implemented in this work to extend the scope of an on-going research project, funded by the Singapore Cancer Syndicate to investigate the use of OCT for early nasopharyngeal cancer (NPC) diagnosis. The first OCT system is designed around a 1300 nm broadband superluminescence diode (SLD) source using the time-domain method. An axial resolution of 10 μm and an imaging depth of more than 2.5 mm were obtained with this system. An existing time-domain 800 nm OCT system based on a Ti:Sapphire laser source is modified into a Fourier-domain system using a nearinfrared (NIR) spectrometer. This eliminates the need for mechanical scanning on the reference arm of the OCT system and improves the imaging time by a factor of 3.7.

Atherosclerosis involves the deposition of lipids in the inner lining of arteries. The resulting build-up is known as plaque. Excessive plaque build-up gives rise to the narrowing of lumens of arteries and is the main cause of disability and death from heart diseases, cerebral infarctions and ischaemia of the limbs. Fatty streaks, or slightly raised yellowish areas, are almost invariably present in the aortas but their presence and extent do not generally correlate with future development of atherosclerosis. On the other hand, the incidence and extent of fatty streaks in the coronary arteries can represent the early stage of this disease. Hence, early detection of plaque in the coronary arteries is important, as the knowledge will allow preventive measures to be administered and inhibit the onset of atherosclerosis. Master of Engineering

Interferometric techniques have been used as imaging techniques in many applications. In using low coherence sources in interferometry, good axial resolution can be achieved. The objective of this thesis is to investigate the use low coherence interferometry in biomedical applications. Low coherence interferometry (LCI) was incorporated into an optical tweezers system to determine the trapping dynamics. An optical coherence tomography (OCT) system based on LCI principles was designed and implemented to detect virus infection in orchid leaves. The OCT system was then modified to realize a high-speed endoscopic OCT imaging system. A numerical method and a common-path probe design were investigated to address the issue of loss of axial resolution due to dispersion. Optical tweezers typically use back-focal interferometry to determine the position of an object in the optical trap. Extensive initial calibration is needed before accurate positional measurements can be made. A LCI system, on the other hand, can provide accurate axial measurements with minimal calibration. A common-path LCI system was incorporated into an optical tweezers system. The light beams of LCI and optical tweezers are collinear, thereby ensuring a good axial accuracy. The trapping dynamics of a trapped microsphere under different trapping conditions were investigated. The results demonstrated that LCI is a viable alternative to back-focal plane interferometry in determining the trapping dynamics. A spectral domain optical coherence tomography (SD-OCT) system was designed and built to identify virus infection in orchid plants. Besides revealing the cross-sectional structure of orchid leaves, a highly scattering upper leaf epidermides were detected with OCT for virus-infected plants. This distinct feature is not observable under histological examination of the leaf samples. The results suggest that virus-infected orchid plants can be accurately identified by imaging the epidermal layers of its leaves with OCT, thereby potentially leading to a better control on the spread of viruses in the orchid industry. In vivo imaging of human tissues has given valuable insights into the understanding of diseases and their management. Conventional in vivo imaging involves white-light endoscopy which relays the optical image of tissue surface back to the user. By contrast, OCT allows the imaging of tissue a few millimeters below the tissue surface which provides additional information on the tissue structure. A gradient-index (GRIN) rod probe was designed and incorporated into a nasopharyngeal rigid probe for both OCT and white-light imaging. To capture an image with minimal motion blur, a fast spectrometer was designed with high spectral resolution. The results showed that there is sufficient resolution in the overall endoscopic OCT probe system to pick out sweat ducts beneath a human skin. The axial resolution of the OCT system depends on the bandwidth of the light source. However, dispersion mismatches between the sample and reference arms of the interferometer broadens the axial point spread function which worsens the axial resolution. A numerical technique was used to extract the phase of the interferogram which is then applied to offset any systemic dispersion mismatches in the system. The results showed that the numerical technique is effective in recovering the OCT image with dispersion. A common path probe with an axicon tip was also proposed to combat dispersion. The axicon tip allowed imaging of a few millimeters without significant loss of resolution due to the diffraction-free properties. The common-path design minimized dispersion and polarization mismatches. OCT images with good axial resolution and imaging depth were obtained with this probe. DOCTOR OF PHILOSOPHY (EEE)

Due to its excellent signal uniformity and reproducibility, well-ordered plasmonic nano-arrays demonstrate great potential as practical SERS probe for real-time in-situ analysis of molecules. A basic study of SERS mechanism was carried out to elucidate relevant factors that can affect SERS enhancement. The results revealed that the rarely discussed scattering cross-section can have substantial influence on SERS enhancement and should be considered for a complete understanding and optimization of the performance of SERS substrates. Two commonly used metallization techniques, DC sputtering and electron-beam (E-beam) evaporation, was investigated to quantify their effect on SERS performance. It was found that the E-beam evaporation technique produces inherently roughened gold film which serve as additional EM hotspots favourable for SERS. A hybrid metal deposition approach to exploit the advantages of both the sputtering and E-beam technique is proposed and verified. Two well-ordered and cost-effective SERS substrates were proposed and investigated. The first is a Au-SiO2 nano-pillar substrate fabricated by performing an additional plasma enhanced chemical vapour deposition of SiO2 on nanosphere-lithorgraphy fabricated Si pillars to tune the plasmonic resonance of the nano-structures. The optimized Au-SiO2 nano-pillar exhibits comparable SERS performance as the well-established gold-film-over-nanosphere substrate and has the added advantage of better stability and reusability due to its inherently rigid structure. The second SERS substrate developed in this work comprises non-lithographic anodized aluminium oxide (AAO)-based nano-fibres (ANF) that are fabricated by optimizing the second anodization and incorporating an additional chemical wet-etching step. The optimized ANF substrate exhibits better sensitivity, signal-to-noise ratio, signal uniformity and stability than those of conventional substrates. The fabrication process of the ANF SERS substrate is also highly reproducible and scalable. Label-free detection of two DNA and RNA nucleobases are also demonstrated with the ANF SERS substrate. Doctor of Philosophy (EEE)

In this thesis, efforts have been made to investigate and optimize the aqueous synthesis route in order to prepare high quality QDs. Reaction for high quality CdTe QD preparation was firstly investigated. It was found that, species and concentration of surfactants are important factors which greatly influence the growth dynamics, optical properties and stability of the QDs. A mix-ligands system composed of mercaptopropionic acid (MPA) and Cysteine (Cys) was then optimized to generate high quality CdTe QDs. To avoid the heavy metal related toxicity issues, high quality Mn-doped ZnSe QDs (d-dots) were synthesized as an alternative to Cd-based QDs. The d-dots are characterized with large Stokes shift and long PL lifetime, and found to be biocompatible for biological applications. A sandwiched core/shell QD (SQD, Mn:ZnSe/ZnS/ZnMnS) was also formulated as high quality contrast agent for both optical fluorescence imaging and magnetic resonance imaging (MRI). After proper surface modification and bio-functionalziation, the d-dots were developed as theranostic agents for in vitro cancer diagnosis and therapy. Two types of nanocarriers, namely d-dot/polymer nanoplex and d-dot/liposome hybrid, were developed as optically traceable nanocarrier for cancer cell targeted gene delivery. The therapeutic effect was confirmed by the significantly suppressed expression of the targeted gene sequence (mutant K-Ras) at mRNA level. The SQD/liposome hybrid was also developed as a multifunctional platform for MR/optical dual mode imaging and gene/drug co-delivery. DOCTOR OF PHILOSOPHY (EEE)