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- W2020005891 abstract "Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the localenvironment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or revealdifferent conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standardlaser 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 todetect fluorescence resonance energy transfer (FRET) in living cells. The results show that different FRET efficienciescan be spatially resolved within a single cell. Furthermore, the upgrade kit does not only allow tomeasure FRET by observing the shortening of the donor lifetime, but also the acceptor decay can be simultaneouslymonitored 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 theprovided 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 LifetimeCorrelation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to removethe influence of detector afterpulsing, which is classically done by cross correlation between two detectors." @default.
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- W2020005891 date "2008-02-07" @default.
- W2020005891 modified "2023-10-18" @default.
- W2020005891 title "Advanced FRET and FCS measurements with laser scanning microscopes based on time-resolved techniques" @default.
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- W2020005891 doi "https://doi.org/10.1117/12.761141" @default.
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