The design of fluorescence correlation spectroscopy for single molecule techniques

Abstract

The ribosome is responsible for protein synthesis- a critical process in creating essential proteins for cell survival. Though bulk techniques yield valuable results about the structure of the ribosome, bulk techniques are not ideal in examining ribosomal dynamics and understanding kinetics involved in protein synthesis. On the contrary, the single molecule spectroscopy techniques are ideal in investigating the mechanisms of ribosome dynamics in real-time under equilibrium conditions. The latest advances in single molecule biophysics have opened numerous opportunities in the biological world to study the dynamics of molecules in real-time. Single molecule techniques such as Fluorescence Correlation Spectroscopy (FCS) have opened opportunities to study the ribosome as well as other ribosomal proteins. FCS is used to investigate diffusion coefficients, concentration, and kinetics of biological samples. In this thesis, we address the process of assembling an FCS system, as well as the design of a high-speed correlator. The high-speed correlator allows the software to handle the high data counts that can occur in single molecule experiments. A field-programmable gate arrays (FPGA) dual correlator and a multi-tau correlator are designed to handle the noise and high count rates that can dominate the signal. Also addressed in this thesis are ideal experimental conditions for successfully obtaining and troubleshooting results received from FCS curves.