Fluorescence correlation spectroscopy (FCS) has evolved to a valuable tool for biomolecular analysis on the single molecule level. Measurements on a single molecule level can only be performed if the measurement volume is small enough to contain on average only very few molecules. Common FCS-systems are therefore based on a confocal
geometry in which a laser spot is focused into a liquid sample. This illumination concept in combination with a pinhole in the detection path leads to an observation volume in the order of one femtoliter. On the other hand, many biomolecular interactions need to be measured on surfaces. To study such interactions or the fluctuating
signal of surface bound molecules itself, as for instance during single molecule enzyme catalysis, evanescent field based excitation seems advantageous as compared to confocal FCS. We discuss different schemes for evanescent field FCS and present an efficient excitation-detection scheme in an objective-based TIR-FCS configuration.
Monitoring biological relevant reactions on the single molecule level by the use of fluorescent probes has become one of the most promising approaches for understanding a variety of phenomena in living organisms. By applying techniques of fluorescence spectroscopy to labelled molecules a manifold of different parameters becomes accessible i.e. molecular dynamics, energy transfer, DNA fingerprinting, etc... can be monitored at the molecular level.
However, many of these optical methods rely on oversimplified assumptions, for example a three-dimensional Gaussian observation volume, perfect overlap volume for different wavelength, etc. which are not valid approximations under many common measurement conditions. As a result, these measurements will contain significant, systematic artifacts, which limit their performance and information content.
Based on Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Spectroscopy we will present representative examples including a thorough signal analysis with a strong emphasis on the underlying optical principles and limitations. An outlook to biochip applications, parallel FCS and parallel Lifetime measurements will be given with cross links to optical concepts and technologies used in industrial inspection.