Applications of single molecule fluorescence detection

Single pair fluorescent resonance energy transfer (spFRET) was used to monitor the conformational dynamics of individual DNA hairpins both free in diffusion and attached to a glass surface. Exponential distributions were found for the native and denatured state lifetimes, which are expected in the simple two-state model of a DNA hairpin. For hairpins with a loop size of forty adenines and a stem size of either seven or nine bases the respective closed-state lifetimes are 45 (+/-2.4) ms and 103 (+/-6.0) ms while the respective open-state lifetimes are 133 (+/-5.5) ms and 142 (+/-22) ms. As predicted, the closed-state lifetime is dependent on the stem length and is independent of the loop characteristics. The measured native and denatured state lifetimes are larger than the numbers expected by extrapolation from a previous study. The bio-specific and chemoselective ligation methods used to immobilize DNA hairpins and other biological macromolecules to glass surfaces, along with both gel and polyacrylamide immobilization methods, are described in detail.;Ratiometric detection of seminaphthorhodafluor (SNARF) fluorophores was used to examine local environmental effects and changes in pH on the single molecule level. SNARF also served as a simple two-state system to examine the distribution of properties of a population on a single molecule level. The protonation-deprotonation process for this fluorophore is shown to be diffusion-limited and evidence for observations of individual protonation events is presented.;Oxygen scavenging systems were examined for their utilities in the prevention of photobleaching of the fluorescent molecules rhodamine 6G (R6G) and fluorescein as well as the tetramethylrhodamine (TMR) and cyanine-5 (Cy5) dyes used in DNA hairpin spFRET measurements. Fluorescence correlation spectroscopy (FCS) was employed to examine the effects of four oxygen scavengers, glucose oxidase (GOD), Trolox(TM), propyl gallate (PG), and mercaptoethylamine (MEA) on the triplet states and photobleaching of R6G and fluorescein. A figure of merit was employed to determine the overall effectiveness of the oxygen scavengers in producing the maximum emission from fluorescent molecules.