title: Pulsed interleaved excitation.
abstract: In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse is complete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements with complexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, where complexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor.
authors.affiliation: Physical Chemistry, Department of Chemistry and Biochemistry, and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandstrasse 11 Haus E, D-81377 Munich, Germany.
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Müller
authors.forename: Evgeny
authors.initials: E
authors.lastname: Zaychikov
authors.forename: Christoph
authors.initials: C
authors.lastname: Bräuchle
authors.forename: Don C
authors.initials: DC
authors.lastname: Lamb
mesh_terms: D001703:Biophysics / Q000295:instrumentation / Q000379:methods*; D002138:Calibration; D002232:Carbocyanines / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D015233:Models, Statistical; D038761:Photobleaching; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D013997:Time Factors; D014162:Transfection
chemical_list: D002232:Carbocyanines; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C085321:cyanine dye 5; D049452:Green Fluorescent Proteins; D004247:DNA