Fluorescence correlation spectroscopy, photophysical aspects and applications

Abstract

Fluorescence correlation spectroscopy (FCS) is a technique where dynamic processes on the molecular level are studied by the use of fluorescence. The molecules are excited within a focused stationary laser beam and the resulting fluorescence fluctuations are analyzed in the form of an autocorrelation function. The autocorrelation function multiplies the fluorescence intensity at a certain time to that theta seconds later. In FCS the autocorrelation function states the probability that a molecule emits a photon at a time theta given an emission at time zero from the same molecule. In principle, information can be obtained about any dynamic process in the nanosecond time range and longer that manifests itself as a change in measured fluorescence intensity.

In this thesis an improved version of the original method is presented. It is based on an extremely small sample volume element from which fluorescence is collected. Due to a strongly reduced background level the sensitivity is so good that single molecule event scan be observed. The sensitivity limits of this method, and of fluorescence spectroscopy in general, is to a large extent determined by photophysical aspects. The total amount and rate of fluorescence emitted per molecule are important figures of merit. They are to a considerable part determined by the tendency of the molecules to undergo intersystem crossing to their triplet states. For most fluorophores the triplet states are non-luminescent and photobleaching of fluorescent molecules are in many cases believed to be proportional to the triplet state population. Here, it is shown how transitions between the triplet and singlet states influence the measured fluorescence autocorrelation functions. It is found that triplet state kinetics can be conveniently measured by FCS. Additionally, the high environmental sensitivity of the triplet state parameters indicates that FCS can be used as a way of probing molecular microenvironments.

With a highly focused laser beam most fluorescent molecules in aqueous solution do not photo bleach within their passage through the beam. However, with an increased beam cross section it is possible to see the photobleaching as a drop in fluorescence already before the molecules exit the laser beam. This makes it possible to analyze the photobleaching. Since many questions still remain open about the origin of photobleaching and since it is thought to be related to the extent of triplet state population the possibility to simultaneously monitor bleaching and triplet state population is very useful.

In order to investigate its potential the present version of FCS was applied to cell surface and chemical kinetics studies. For diffusion measurements of proteins in cell membranes FCS has previously not found any applications. However, with the higher sensitivity and the higher relative fluctuations in fluorescence that can be measured with the present setup it is likely that FCS can be a complement to the more frequently used technique of fluorescence photobleaching recovery (FPR). In the chemical kinetics section different inter- and intramolecular processes are monitored, including binding of alpha-bungarotoxin to the nicotinic Acetylcholine receptor, interaction of Protein Kinase C with lipids, binding of calcium and hydrogen ions to ion-sensitive fluorescent indicators, base-specific dye-nucleotide interactions and the trans-cis isomerization of a cyanine dye. The association and dissociation rate constants span over 7 and 11 orders of magnitude, respectively. This illustrates the wide applicability and the wide time range over which dynamical processes can be monitored by FCS.