New Applications of Pulsed Interleaved Excitation

Abstract

Several years ago, the group of Simon Weiss introduced the concept of alternating laser excitation, which we combined with subnanosecond pulsed lasers in the method of pulsed interleaved excitation (PIE). In PIE, it is possible to determine which excitation pulse is responsible for generating the detected photon. With this additional piece of information, one can, for example, separate donor-only and acceptor-only species from double-labeled species in single-pair Forster Resonance Energy Transfer (spFRET) experiments, remove spectral cross-talk in fluorescence cross-correlation spectroscopy (FCCS) or use it to determine FRET efficiencies from an FCCS experiment.We have now combined PIE with scanning fluorescence correlation spectroscopy (FCS) and with raster image correlation spectroscopy (RICS). Both scanning FCS and RICS are preferable for fluctuation measurements in living cells where diffusion is slowed and photobleaching is an issue. By scanning, several volumes can be measured in parallel, improving statistics and minimizing photobleaching. However, spectral crosstalk can still be a limiting factor for these experiments, especially when using autofluorescent fusion proteins. Here, we demonstrate the ability of PIE to remove spectral crosstalk for these scanning methods, making scanning FCCS and cross-correlation RICS more sensitive to interactions.It is also possible to combine PIE with multiparameter fluorescence detection (MFD) to perform highly accurate spFRET experiments. When performing spFRET experiments, there are many factors that can limit the accuracy of the experiment. These include correction for spectral crosstalk, direct excitation of the acceptor, photobleaching during a burst, and uncertainties in the orientation factor. To correct for all these issues, often multiple experiments are performed. We demonstrate that, using the capability of PIE and MFD to separate different species, all parameters necessary for performing an accurate spFRET experiment can be obtained in a single measurement.