Multi-Parameter Fluorescence Detection at the Single-Molecule Level : Techniques and Applications

Abstract

Fluorescence-based assays using single-molecule techniques are evolving into a very important tool in science. These techniques include not only direct detection and analysis of single-molecule events, but also spectroscopic analysis through fluctuation methods such as FCS (fluorescence correlation spectroscopy) or FIDA (fluorescence intensity distribution analysis). What are the advantages of these techniques and why is fluorescence chosen as the readout? This article will give an overview of fluorescence techniques and applications of single-molecule detection and analysis. It is focused on the detection of fluorescence emitted from freely diffusing molecules in solution. A confocal microscope is described, which uses special electronics for detection and enables the observation of fluorescence emission from single molecules along with monitoring of various fluorescence parameters (MFD). In the first part, procedures such as the detection and identification of single molecules, the monitoring of rare events, the direct temporal observation of binding events and conformational dynamics, as well as the possibility of selective spectroscopy of molecular states are described. Industrial applications such as HTS are illustrated by a description of using fluctuation methods such as FCS and FIDA to resolve characteristic properties of biological assays, e.g., ligand-protein and -vesicle binding or enzymatic reactions.