Single-molecule tracking and its application in biomolecular binding detection
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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.