Single-molecule tracking and its application in biomolecular binding detection

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. While 3D-SMT is probably the most suitable method for determining whether tracked molecules (can be any biomolecule such as DNA, membrane receptors, and transcription factors) form dimers or complexes with other molecules, great technical challenges remain to be overcome before the potential of 3D-SMT in biomolecular binding detection can be realized. This dissertation describes my work on recent methodology development to overcome these challenges, and new applications of the 3D-SMT technology in rare molecular species quantification. First, we provide an overview of current SMT technologies, with an emphasis on three-dimensional feedback controlled SMT. Advantages and drawbacks of each SMT method are outlined. Second, we describe the theoretical modeling and instrumentation of our confocal tracking microscope. Its multi-dimensional sensing capability (3D position, diffusion coefficient, fluorescence lifetime) is experimentally characterized. In order to maximize the tracking duration, we have also developed strategies to effectively slow-down fast diffusing molecule, and optimized the buffer conditions. Third, we show that our 3D-SMT microscope can detect biomolecular association/disassociated by two types of contrast mechanisms: diffusion rate and lifetime FRET signal. DNA transient binding is used as a model system because of ease of fluorescent labeling and tunable binding kinetics. Both of the two mechanisms involve tracking a fluorescent-labeled single-stranded DNA (ssDNA), but the second approach also requires its complementary strand to be labeled by a dark quencher. A combined analysis of multiple single-molecule trajectories allow us to measure the kinetics that is even beyond the physical bandwidth of the tracking system. In the end, we introduce the application of SMT in rare single-molecule species quantification. The theory for predicting the sensitivity and fidelity is established. Our work highlights the fundamental limitations that we are facing in precise single-molecule identification and quantification without amplification.Biomedical Engineerin

Fano resonance engineering in mirror-symmetry-broken THz metamaterials

We introduce a comprehensive approach to the design of mirror-symmetry broken terahertz (THz) metamaterials and present both the simulation and experimental results which show the desired asymmetric Fano resonances and electromagnetic induced transparency (EIT)-like windows. With a full wave simulation, we find these asymmetry-induced resonance modes possess extremely high quality factors and they broaden with increase of the structure asymmetry. This phenomenon arises from the destructive interference of a super-radiative bright mode and a sub-radiative dark mode which can’t be excited directly. Surface current and electric field distributions are analyzed to explain the emergence of these Fano resonances. An intuitive mechanical coupled oscillator model is derived to explain the unique line-shape of such Fano resonances. Moreover, large resonant frequency tuning (50 GHz) of Fano resonance has been demonstrated by temperature induced phase change in liquid crystals. We believe that the Fano resonance in THz metamaterials may serve as a strong building block for passive or active THz elements with potential applications for future detection and sensing systems and devices.The authors would like to thank the UK Engineering and Physical Sciences Research Council (EPSRC) for the support through the Platform Grant for Liquid Crystal Photonics (EP/F00897X/1). Xuefeng Li would like to acknowledge the support from Cambridge Trust.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Springer

Detection of copy number variations in rice using array-based comparative genomic hybridization

<p>Abstract</p> <p>Background</p> <p>Copy number variations (CNVs) can create new genes, change gene dosage, reshape gene structures, and modify elements regulating gene expression. As with all types of genetic variation, CNVs may influence phenotypic variation and gene expression. CNVs are thus considered major sources of genetic variation. Little is known, however, about their contribution to genetic variation in rice.</p> <p>Results</p> <p>To detect CNVs, we used a set of NimbleGen whole-genome comparative genomic hybridization arrays containing 718,256 oligonucleotide probes with a median probe spacing of 500 bp. We compiled a high-resolution map of CNVs in the rice genome, showing 641 CNVs between the genomes of the rice cultivars 'Nipponbare' (from <it>O. sativa </it>ssp. <it>japonica</it>) and 'Guang-lu-ai 4' (from <it>O. sativa </it>ssp. <it>indica</it>). The CNVs identified vary in size from 1.1 kb to 180.7 kb, and encompass approximately 7.6 Mb of the rice genome. The largest regions showing copy gain and loss are of 37.4 kb on chromosome 4, and 180.7 kb on chromosome 8. In addition, 85 DNA segments were identified, including some genic sequences. Contracted genes greatly outnumbered duplicated ones. Many of the contracted genes corresponded to either the same genes or genes involved in the same biological processes; this was also the case for genes involved in disease and defense.</p> <p>Conclusion</p> <p>We detected CNVs in rice by array-based comparative genomic hybridization. These CNVs contain known genes. Further discussion of CNVs is important, as they are linked to variation among rice varieties, and are likely to contribute to subspecific characteristics.</p

Evidence-based PET for thoracic tumours

AbstractFluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) is a robust imaging tool that is currently used in daily clinical practice for the evaluation of thoracic malignancies. This chapter provides an overview of the current evidence-based data on the usefulness of PET/CT for the evaluation of patients with thoracic tumours including lung cancer, pleural and thymic tumours, and esophageal cancer