Laser-induced fluorescence detection of single fluorescent molecules represents the ultimate level of sensitivity for fluorescence-based assays in analytical chemistry, biology, and medicine (1-14). Various groups have achieved singlemolecule detection in solution (1-6) and at surfaces (7-10).Recently it was possible to characterize single molecules in solution by their fluorescence emission spectra (12) or fluorescence lifetimes (13,14). Single-molecule spectroscopy eliminates ensemble averaging and allows individual members of a population to be probed, thereby yielding more direct information regarding the distribution of molecular and kinetic properties. This constitutes a major step toward the study of single-molecule dynamics and molecular recognition in solution. The recent improvements in single-molecule detection lead us to expect many diagnostic applications for such measurement techniques in the near future, such as monitoring enzyme function and conformational dynamics of DNA.An increasing number of publications (15-19) employing fluorophores covalently linked to polynucleotide sites illustrate the widespread interest in investigating thermodynamic, kinetic, and structural characteristics of various DNA molecules. Yet the fluctuations of the conjugated fluorophore between several conformational states, each with a different fluorescence quantum yield (15, 16), introduces a large degree of uncertainty to such evaluations.Rigler and coworkers recently investigated the structural dynamics measurements of a tetramethylrhodamine (TMR)-labeled DNA duplex in solution (16) and immobilized on a surface (20). They performed classical ensemble and singlemolecule measurements using fluorescence correlation and time-resolved fluorescence spectroscopy that were completed by time-integrated fluorescence decay measurements of single-molecule transits. These authors proposed a millisecond equilibrium between two conformational states characterized by different photobleaching lifetimes, fluorescence lifetimes of 0.9 and 3.7 ns, and a widely distributed conformational relaxation rate constant obeying a stretched exponential law, which indicates the existence of numerous conformational substates (21).So far, conventional confocal spectroscopic techniques (1, 16) have been used to analyze several fluorescence bursts caused by individual molecules diffusing through the microscopic, open measurement volume at once, thereby resulting in an averaging of properties over the single-molecule population under analysis. Fluorescence decay curves have been measured for single molecules (16,22), but the signal was integrated over the entire observation time, resulting in a loss of the kinetic information. Therefore, we have developed an experimental technique for specific burst analysis that avoids the inclusion of extraneous events and registers the macroscopic detection time of each single photon event for subsequent kinetic analysis.In this paper, we describe the identification and detection of different temporally resolved conf...
Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) are introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over the detection volume together with saturation effects of the fluorescence and of the detection electronics, the probability of different transit times through the detection volume, and the probability of multimolecule events. Using BIFL together with selective counting, the concentration of two dyes, Rhodamine B and Rhodamine 6G, in separate solutions and in a mixture were determined. The obtained results are consistent with the applied dye concentrations and with simultaneous measurements by fluorescence correlation spectroscopy (FCS). The introduced method is an appropriate tool for the complete characterization and quantitative analysis of a highly diluted sample in homogeneous assays.
Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F I part of the H + -ATPase from Escherichia coli, EF I , carrying the Q QT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355^21359] in aqueous solution upon excitation with a modelocked argon ion laser at 528 nm. The diffusion of the labeled EF I through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3U10^U cm P s^I. In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF I molecules allows the detection of conformational changes.z 1998 Federation of European Biochemical Societies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.