Understanding of Förster Resonance Energy Transfer (FRET) in Ionic Materials

Herein, an ionic material (IM) with Förster Resonance Energy Transfer (FRET) characteristics is reported for the first time. The IM is designed by pairing a Nile Blue A cation (NBA+) with an anionic near-infrared (NIR) dye, IR820−, using a facile ion exchange reaction. These two dyes absorb at different wavelength regions. In addition, NBA+ fluorescence emission spectrum overlaps with IR820− absorption spectrum, which is one requirement for the occurrence of the FRET phenomenon. Therefore, the photophysical properties of the IM were studied in detail to investigate the FRET mechanism in IM for potential dye sensitized solar cell (DSSCs) application. Detailed examination of photophysical properties of parent compounds, a mixture of the parent compounds, and the IM revealed that the IM exhibits FRET characteristics, but not the mixture of two dyes. The presence of spectator counterion in the mixture hindered the FRET mechanism while in the IM, both dyes are in close proximity as an ion pair, thus exhibiting FRET. All FRET parameters such as spectral overlap integral, Förster distance, and FRET energy confirm the FRET characteristics of the IM. This article presents a simple synthesis of a compound with FRET properties which can be further used for a variety of applications.

Download Full-text

A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission

Journal of The Royal Society Interface ◽

10.1098/rsif.2008.0381.focus ◽

2008 ◽

Vol 6 (suppl_1) ◽

Cited By ~ 68

Author(s):

A.D Elder ◽

A Domin ◽

G.S Kaminski Schierle ◽

C Lindon ◽

J Pines ◽

...

Keyword(s):

Energy Transfer ◽

Protein Interactions ◽

Imaging Techniques ◽

Resonance Energy Transfer ◽

Fluorescence Emission ◽

Resonance Energy ◽

Forster Resonance Energy Transfer ◽

Confocal Imaging ◽

Förster Resonance Energy Transfer ◽

Förster Resonance

Fluorescence detection of acceptor molecules sensitized by Förster resonance energy transfer (FRET) is a powerful method to study protein interactions in living cells. The method requires correction for donor spectral bleed-through and acceptor cross-excitation as well as the correct normalization of signals to account for varying fluorophore concentrations and imaging parameters. In this paper, we review different methods for FRET signal normalization and then present a rigorous model for sensitized emission measurements, which is both intuitive to understand and practical to apply. The method is validated by comparison with the acceptor photobleaching and donor lifetime-imaging techniques in live cell samples containing EYFP and ECFP tandem constructs exhibiting known amounts of FRET. By varying the stoichiometry of interaction in a controlled fashion, we show that information on the fractions of interacting donors and acceptors can be recovered. Furthermore, the method is tested by performing measurements on different microscopy platforms in both widefield and confocal imaging modes to show that signals recovered under different imaging conditions are in quantitative agreement. Finally, the method is applied in the study of dynamic interactions in the cyclin–cdk family of proteins in live cells. By normalizing the obtained signals for both acceptor and donor concentrations and using a FRET exhibiting control construct for calibration, stoichiometric changes in these interactions could be visualized in real time. The paper is written to be of practical use to researchers interested in performing sensitized emission measurements. The correct interpretation of the retrieved signals in a biological context is emphasized, and guidelines are given for the practical application of the developed algorithms.

Download Full-text