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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Müller BK,Zaychikov E,Bräuchle C,Lamb DC

doi

10.1529/biophysj.105.064766

subject

Has Abstract

pub_date

2005-11-01 00:00:00

pages

3508-22

issue

5

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)72991-X

journal_volume

89

pub_type

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