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Biophys. J. BioFAST: First Published August 19, 2005. doi:10.1529/biophysj.105.064766
© 2005 by the Biophysical Society.

A more recent version of this article appeared on November 1, 2005.
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SPECTROSCOPY, IMAGING, OTHER TECHNIQUES

Pulsed Interleaved Excitation

Barbara K. Müller 1, Evgeny Zaychikov 2, Christoph Bräuchle 1 and Don C. Lamb 3*

1 Ludwig-Maximilians-Universität München
2 Max-Planck Institute for Biochemistry
3 LMU Munich

* To whom correspondence should be addressed. E-mail: don.lamb{at}cup.uni-muenchen.de.

Submitted on April 17, 2005
Revised on May 18, 2005
Accepted on 9 August 2005


  Abstract
In this paper, 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 circa 1 MHz 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 multi-color 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.

Key Words: Alternating Laser Excitation, Confocal Microscopy, Fluorescence Correlation Spectroscopy, Fluorescence Resonance Energy Transfer, Pulsed Interleaved Excitation




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Copyright © 2005 by the Biophysical Society.