Spectral analysis in microscopy : a study of FRET and single quantum dot luminescence

Abstract

This thesis deals with the development of new techniques and luminescent markers, to improve the quality of luminescence studies in microscopy. A sensitive spectrograph that can be used for spectrally resolved emission spectroscopy in the microscope is described, including design considerations, specifications and applications. The spectrograph is applied to spectral imaging studies of Förster Resonance Energy Transfer (FRET) and the study the photo-physical properties of fluorescence dyes and enhanced mutants of the Green Fluorescent Proteins. Furthermore, the spectrograph is applied to study the luminescence properties of single quantum dots by monitoring the luminescence spectrum of microvolumes with millisecond time-resolution. Part of this thesis describes an analysis method that uses Monte Carlo Simulations to describe the temporal and spatial variations of the fluorescence intensity in a microscopic sample that exhibits FRET, but suffers from photobleaching. Furthermore the Monte Carlo simulations were applied to verify the suitability of a more simple analysis method to calculate the FRET efficiency.