Fluorescence correlation spectroscopy, photophysical aspects and applications

Sammanfattning: Fluorescence correlation spectroscopy (FCS) is a technique where dynamic processes onthe molecular level are studied by the use of fluorescence. The molecules are excitedwithin a focused stationary laser beam and the resulting fluorescence fluctuations areanalyzed in the form of an autocorrelation function. The autocorrelation functionmultiplies the fluorescence intensity at a certain time to that theta seconds later. In FCS theautocorrelation function states the probability that a molecule emits a photon at a time thetagiven an emission at time zero from the same molecule. In principle, information can beobtained about any dynamic process in the nanosecond time range and longer thatmanifests itself as a change in measured fluorescence intensity. In this thesis an improved version of the original method is presented. It is based on anextremely small sample volume element from which fluorescence is collected. Due to astrongly reduced background level the sensitivity is so good that single molecule eventscan be observed. The sensitivity limits of this method, and of fluorescence spectroscopy in general, is to alarge extent determined by photophysical aspects. The total amount and rate offluorescence emitted per molecule are important figures of merit. They are to aconsiderable part determined by the tendency of the molecules to undergo intersystemcrossing to their triplet states. For most fluorophores the triplet states are nonluminescentand photobleaching of fluorescent molecules are in many cases believed to be proportionalto the triplet state population. Here, it is shown how transitions between the triplet andsinglet states influence the measured fluorescence autocorrelation functions. It is foundthat triplet state kinetics can be conveniently measured by FCS. Additionally, the highenvironmental sensitivity of the triplet state parameters indicates that FCS can be used as away of probing molecular microenvironments. With a highly focused laser beam mostfluorescent molecules in aqueous solution do not photobleach within their passage throughthe beam. However, with an increased beam cross section it is possible to see thephotobleaching 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 remainopen about the origin of photobleaching and since it is thought to be related to the extentof triplet state population the possibility to simultaneously monitor bleaching and tripletstate population is very useful. In order to investigate its potential the present version of FCS was applied to cellsurface and chemical kinetics studies. For diffusion measurements of proteins in cellmembranes FCS has previously not found any applications. However, with the highersensitivity and the higher relative fluctuations in fluorescence that can be measured withthe present setup it is likely that FCS can be a complement to the more frequently usedtechnique of fluorescence photobleaching recovery (FPR). In the chemical kinetics sectiondifferent inter- and intramolecular processes are monitored, including binding of alpha-bungarotoxin to the nicotinic Acetylcholine receptor, interaction of Protein Kinase C withlipids, 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. Theassociation and dissociation rate constants span over 7 and 11 orders of magnitude,respectively. This illustrates the wide applicability and the wide time range over whichdynamical processes can be monitored by FCS.