In situ optically trapped probing system for molecular recognition and localization.

摘要

This research dissertation presents design and implementation of a complete multi-functional optically-trapped probing system for in-situ molecular recognition and localization. This complete system consists of three-axis steering system, dual-trap manipulation, and dual-laser detection with advanced implementation of automated sample-probe engagement by high-speed confocal imaging and real-time calibration and force probing.;The development of the optically-trapped probing system was based on the knowledge and expertise acquired from two bench-test systems. The first system was built with a three-axis lens translation system to manually steer the trapping laser. The second system successfully implemented the back-focal-plane (BFP) interferometry for position measurement to complete the system with the capabilities of three-dimensional (3D) manipulation, position detection, and force sensing with a stationary optical trap. The integration mechanism of rapid three-axis laser steering is able to achieve over 50 kHz bandwidth laterally with a two-axis acousto-optic deflector (AOD) and over 3 kHz vertically with a deformable mirror (DM) by model cancellation. Two trapping lasers and one measurement laser were well aligned to achieve dual-trap manipulation and dual-laser measurement for relative and absolute displacement of the probe.;Real-time in situ calibration for measurement sensitivity and trapping stiffness was illustrated based on the weighted recursive least square (WRLS) algorithm with the system parameters for time-varying optical trapping system, such as the state transition coefficient, recursively estimated according to the probe's motion. This algorithm has been successfully applied to calibrate the measurement sensitivity and trapping stiffness and is applicable for probe's local temperature estimation with slight modification to the algorithm. Two experiments were conducted to demonstrate the capability of this realtime calibration algorithm in response to the variation of trapping laser power and environmental temperature.;High-speed confocal imaging technology was intensively investigated and an application-oriented project for automated sample-probe engagement was collaboratively implemented with Dr Peng Cheng. The confocal-imaging based interface is capable of two-dimensional (2D) dynamic tracking and 3D sample-probe engagement with information linked with the optically trapped probing system.;Quasi-static and real-time force probing were encapsulated to study specific/nonspecific interactions between the probe functionalized with M2-antibodies and the cell samples labeled by Flag epitode. The quasi-static force sensing was employed to probe this interaction while the probe and sample approached stepwisely. Specific interaction and non-specific interaction were detected with distinctive results. The real-time force sensing was applied to investigate the process in the continuous trace and retrace with respect to the probe. Experimental results for a Human Embryonic Kidney (HEK) 293 live cell provided intrinsic information of the system dynamics for specific interaction. Position-clamped real-time force probing was successfully employed for 3D force mapping of Flag-tagged beads and HEK 293 live cells expressed with Flag-NIS-eGFP. A few concerns related to practical biological experiments were illustrated.;The main contribution of this research is design and development of the unique multi-functional optically-trapped probing system for in-situ biological applications in quasi-static and real-time force sensing for specific interactions and 3D force mapping for molecular localization.