title: FRET-enabled optical modulation for high sensitivity fluorescence imaging.
abstract: 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).
authors.affiliation: School of Chemistry and Biochemistry and Petit Institute for Biosciences and Bioengineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.
authors.forename: Chris I
authors.initials: CI
authors.lastname: Richards
authors.forename: Jung-Cheng
authors.initials: JC
authors.lastname: Hsiang
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Khalil
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Hull
authors.forename: Robert M
authors.initials: RM
authors.lastname: Dickson
mesh_terms: 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
chemical_list: D002232:Carbocyanines; C089187:cyanine dye 3; C085321:cyanine dye 5; D004247:DNA
references.citation: J Phys Chem B. 2010 Jan 14;114(1):660-5
references.pmid: 19902923
references.citation: Angew Chem Int Ed Engl. 2008;47(29):5465-9
references.pmid: 18601270
references.citation: Bioconjug Chem. 2003 Jan-Feb;14(1):195-204
references.pmid: 12526709
references.citation: Nature. 1997 Jul 24;388(6640):355-8
references.pmid: 9237752
references.citation: J Phys Chem A. 2007 Mar 8;111(9):1593-7
references.pmid: 17295461
references.citation: Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7462-5
references.pmid: 17042053
references.citation: Science. 2007 May 25;316(5828):1191-4
references.pmid: 17525339
references.citation: J Am Chem Soc. 2006 Sep 6;128(35):11423-32
references.pmid: 16939265
references.citation: Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17789-94
references.pmid: 19004775
references.citation: Nat Biotechnol. 2003 Nov;21(11):1387-95
references.pmid: 14595367
references.citation: Eur Biophys J. 2009 Jul;38(6):807-12
references.pmid: 19536533
references.citation: J Phys Chem B. 2006 Nov 23;110(46):23221-7
references.pmid: 17107169
references.citation: J Phys Chem A. 2006 Jan 12;110(1):45-50
references.pmid: 16392838
references.citation: Nat Methods. 2008 Feb;5(2):159-61
references.pmid: 18176568
references.citation: Eur Biophys J. 2008 Nov;38(1):69-82
references.pmid: 18769914
references.citation: J Chem Phys. 2005 Jul 22;123(4):044502
references.pmid: 16095364
references.citation: J Am Chem Soc. 2009 Apr 8;131(13):4619-21
references.pmid: 19284790
references.citation: J Microsc. 2000 May;198(Pt 2):82-7
references.pmid: 10810003
references.citation: Nucleic Acids Res. 1995 May 25;23(10):1795-9
references.pmid: 7784185
references.citation: Opt Lett. 1994 Jun 1;19(11):780-2
references.pmid: 19844443
references.citation: Science. 2007 Sep 21;317(5845):1749-53
references.pmid: 17702910
references.citation: J Am Chem Soc. 2008 Dec 17;130(50):16840-1
references.pmid: 19053449
references.citation: Biophys J. 2008 Jul;95(2):729-37
references.pmid: 18424494
references.citation: Science. 2005 Jun 3;308(5727):1469-72
references.pmid: 15817813
references.citation: J Am Chem Soc. 2009 Apr 15;131(14):5018-9
references.pmid: 19301868
references.citation: Phys Rev Lett. 2007 Nov 30;99(22):228105
references.pmid: 18233334
references.citation: Proc Natl Acad Sci U S A. 2009 May 19;106(20):8107-12
references.pmid: 19433792
references.citation: J Phys Chem B. 2007 Jun 28;111(25):6987-91
references.pmid: 17530801
references.citation: Biophys J. 2006 Dec 1;91(11):4258-72
references.pmid: 16980368
references.citation: Science. 2007 May 25;316(5828):1153-8
references.pmid: 17525330
references.citation: Nat Cell Biol. 2000 Mar;2(3):168-72
references.pmid: 10707088
references.citation: Biophys J. 2008 Jun;94(11):4515-24
references.pmid: 18281383
references.citation: Nat Methods. 2008 Nov;5(11):947-9
references.pmid: 18794860
references.citation: J Am Chem Soc. 2007 Oct 24;129(42):12746-55
references.pmid: 17900110
references.citation: Science. 2006 Sep 15;313(5793):1642-5
references.pmid: 16902090
references.citation: J Am Chem Soc. 2005 Mar 23;127(11):3801-6
references.pmid: 15771514