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Partial Acceptor Photobleaching-Based Quantitative FRET Method Completely Overcoming Emission Spectral Crosstalks

Published online by Cambridge University Press:  02 October 2012

Huali Li ,
Huaina Yu  and
Tongsheng Chen
Huali Li
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Huaina Yu
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
Tongsheng Chen*
Affiliation:
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
*
*Corresponding author. E-mail: [email protected]
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Abstract

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

Keywords

FRETpartial acceptor photobleachingdonor-acceptor pairacceptor emission spectral crosstalkfluorescent proteinmicroscopy
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1021 - 1029
Copyright
Copyright © Microscopy Society of America 2012

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