FRET-enabled optical modulation for high sensitivity fluorescence imaging.
Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield >>10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).
Richards
Chris I
CI
School of Chemistry and Biochemistry and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.
Hsiang
Jung-Cheng
JC
Khalil
Andrew M
AM
Hull
Nathan P
NP
Dickson
Robert M
RM
D001483:Base Sequence; D002232:Carbocyanines / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D055029:Inverted Repeat Sequences; D057054:Molecular Imaging / Q000379:methods*; D055620:Optical Phenomena
D002232:Carbocyanines; C089187:cyanine dye 3; C085321:cyanine dye 5; D004247:DNA
J Phys Chem B. 2010 Jan 14;114(1):660-5
19902923
Angew Chem Int Ed Engl. 2008;47(29):5465-9
18601270
Bioconjug Chem. 2003 Jan-Feb;14(1):195-204
12526709
Nature. 1997 Jul 24;388(6640):355-8
9237752
J Phys Chem A. 2007 Mar 8;111(9):1593-7
17295461
Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7462-5
17042053
Science. 2007 May 25;316(5828):1191-4
17525339
J Am Chem Soc. 2006 Sep 6;128(35):11423-32
16939265
Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17789-94
19004775
Nat Biotechnol. 2003 Nov;21(11):1387-95
14595367
Eur Biophys J. 2009 Jul;38(6):807-12
19536533
J Phys Chem B. 2006 Nov 23;110(46):23221-7
17107169
J Phys Chem A. 2006 Jan 12;110(1):45-50
16392838
Nat Methods. 2008 Feb;5(2):159-61
18176568
Eur Biophys J. 2008 Nov;38(1):69-82
18769914
J Chem Phys. 2005 Jul 22;123(4):044502
16095364
J Am Chem Soc. 2009 Apr 8;131(13):4619-21
19284790
J Microsc. 2000 May;198(Pt 2):82-7
10810003
Nucleic Acids Res. 1995 May 25;23(10):1795-9
7784185
Opt Lett. 1994 Jun 1;19(11):780-2
19844443
Science. 2007 Sep 21;317(5845):1749-53
17702910
J Am Chem Soc. 2008 Dec 17;130(50):16840-1
19053449
Biophys J. 2008 Jul;95(2):729-37
18424494
Science. 2005 Jun 3;308(5727):1469-72
15817813
J Am Chem Soc. 2009 Apr 15;131(14):5018-9
19301868
Phys Rev Lett. 2007 Nov 30;99(22):228105
18233334
Proc Natl Acad Sci U S A. 2009 May 19;106(20):8107-12
19433792
J Phys Chem B. 2007 Jun 28;111(25):6987-91
17530801
Biophys J. 2006 Dec 1;91(11):4258-72
16980368
Science. 2007 May 25;316(5828):1153-8
17525330
Nat Cell Biol. 2000 Mar;2(3):168-72
10707088
Biophys J. 2008 Jun;94(11):4515-24
18281383
Nat Methods. 2008 Nov;5(11):947-9
18794860
J Am Chem Soc. 2007 Oct 24;129(42):12746-55
17900110
Science. 2006 Sep 15;313(5793):1642-5
16902090
J Am Chem Soc. 2005 Mar 23;127(11):3801-6
15771514