Fluorescence correlation spectroscopy

Abstract

Fluorescence Correlation Spectroscopy (FCS) has become an important tool for measuring diffusion, concentration and molecular interactions in biomolecular systems. Two major requirements for FCS are good collection efficiency and a small and well defined excitation volume. This thesis presents an elegant approach in addressing these basic requirements via a single lens element. The Solid Immersion Lens (SIL) is used to obtain a highly confined light field in the excitation volume and also good collection efficiency. The SIL was utilized with a standard microscope objective and the results compared to that of a conventional high NA water immersion objective. With suitable aberration compensation in solution it was proved that the performance with the SIL was similar to that close to the surface. The field characterization for the SILs FCS is described by the vectorial Debye diffraction theory with an emphasis on the aberration function for the SIL with generalized Zernike polynomials. The accurate determination of the collection volume geometry for the SIL is also shown by utilizing the vectorial diffraction theory. The most important advance here was to minimize the illuminated sample volume with increased collection efficiency at increasing focusing depths in the sample with aberration compensation. For many applications in the field of life sciences, biology and medicine a well controlled heating of the sample is necessary. Therefore a thermal, mechanical and/or electrical decoupling is of great interest and utility. This handling modality is easily realized due to the separation of the SIL lens and the microscope objective and/or microscope body. This is demonstrated by the SIL thermal chamber which is used to measure vesicles at varying temperatures. In extension to the work related to FCS in general, the utilization of a novel global stochastic algorithm is studied for analyzing FCS data. This new application would automate FCS data analysis reducing user induced errors to a blackbox level. The theoretical treatment extends to simulations performed on raw photon traces on a software multiple tau correlator and over noise analysis on experimental data.