In this paper we present recent spectroscopic studies using a Solid Immersion Lens for Fluorescent Correlation Spectroscopy measurements. We compare the performance of the Solid Immersion Lens confocal microscope built-up in our group to the performance of a conventional confocal microscope used for FCS. The novelty of the new SIL-FCS microscope is a system containing a conventional objective (NA = 0.6) combined with a Solid Immersion Lens used for single molecule experiment. Important parameters for single molecule experiments such as collection efficiency and excitation field confinement are investigated for different modes of the SIL objective system.
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.