Advanced FRET and FCS measurements with laser scanning microscopes based on time-resolved techniques

Abstract

Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard laser scanning confocal microscopes towards a time-resolved system, which is based on picosecond pulsed lasers, fast detectors and sophisticated single photon counting electronics. We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to detect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET efficiencies can be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow to measure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneously monitored using two spectrally separated detectors and a router. A very special feature of the upgrade kit is that it uses an unrestricted data acquisition approach. With this approach, not only Fluorescence Lifetime Imaging Microscopy (FLIM) with single molecule sensitivity is realized, but the provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy (FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Lifetime Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove the influence of detector afterpulsing, which is classically done by cross correlation between two detectors.