Building a Fluorescence Correlation Spectroscopy (FCS) instrument followed bytesting the boundaries of FRET-FCS

Abstract: Understanding biological systems oftentimes requires mapping the behaviors of biomolecules on small scales such as single proteins or nucleic acids. Fluorescence Correlation Spectroscopy (FCS) is a group of particularly noninvasive and sensitive fluorescence-­based techniques that can be used for this purpose. In this project an FCS-­apparatus has been built and consequently used to test the boundaries of a newly developed variant of FCS called FRET­FCS (Förster Resonance Energy Transfer). Unlike regular FCS, this technique employs fluorescence resonance energy transfer (FRET) in addition to fluorescence. In practice, testing the boundaries means adding increasing amounts of FRET­ DNA to a mix of red­ respectively green marked DNA and determine the lower limit for which FRET­-DNA can be detected. A functioning FCS-­setup was built. When FCS­ and FCCS (Fluorescence Cross­ Correlation Spectroscopy)­ measurements were conducted, the desired curves for the intensity functions were generated. The result of the sensitivity analysis was that the lower limit of detectable FRET­-DNA was approximately 1.3% of a sample containing FRET­-DNA and single­ marked DNA.