title: Pulsed interleaved excitation fluctuation imaging.
abstract: Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.
authors: Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany. jelle_hendrix@hotmail.com.
authors: Jelle
authors: J
authors: Hendrix
authors: Waldemar
authors: W
authors: Schrimpf
authors: Matthias
authors: M
authors: Höller
authors: Don C
authors: DC
authors: Lamb
mesh_terms: D002470:Cell Survival; D003116:Color; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins
references: Chemphyschem. 2012 Mar;13(4):1036-53
references: 22407544
references: Microsc Res Tech. 2006 Nov;69(11):933-9
references: 16941642
references: Biophys J. 2012 Oct 17;103(8):1672-82
references: 23083710
references: Biophys J. 2006 Jul 15;91(2):715-27
references: 16632502
references: Biophys J. 2008 Feb 15;94(4):1437-48
references: 17933881
references: J Fluoresc. 2010 Jan;20(1):105-14
references: 19690947
references: Biophys J. 2006 Sep 1;91(5):1915-24
references: 16782786
references: Biophys J. 2004 Jan;86(1 Pt 1):506-16
references: 14695295
references: Nucleic Acids Res. 2006;34(14):e102
references: 16914444
references: Biophys J. 2011 Apr 6;100(7):1810-8
references: 21463595
references: Biophys J. 2006 Nov 15;91(10):3921-36
references: 16950841
references: Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):151-6
references: 10618386
references: J Microsc. 2008 Jan;229(Pt 1):78-91
references: 18173647
references: J Cell Biol. 1987 Jul;105(1):41-8
references: 3112165
references: Biophys J. 2008 Mar 15;94(6):2320-32
references: 18096627
references: Biophys J. 2005 Nov;89(5):3508-22
references: 16113120
references: Biophys J. 2012 Mar 7;102(5):1174-83
references: 22404940
references: J Fluoresc. 2007 Jan;17(1):43-8
references: 17171439
references: Cytometry. 1999 Jul 1;36(3):176-82
references: 10404965
references: Langmuir. 2009 May 5;25(9):5209-18
references: 19260653
references: J Fluoresc. 2008 Sep;18(5):813-9
references: 18204890
references: Biophys J. 2003 Sep;85(3):1948-58
references: 12944307
references: Chemphyschem. 2012 Mar;13(4):1060-78
references: 22383292
references: Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22287-92
references: 20018714
references: Biophys J. 1993 Sep;65(3):1135-46
references: 8241393
references: Biophys J. 2009 Jan;96(2):707-16
references: 19167315
references: Methods Enzymol. 2013;518:205-43
references: 23276541
references: Nat Biotechnol. 2002 Jan;20(1):87-90
references: 11753368
references: Curr Pharm Biotechnol. 2009 Aug;10(5):543-58
references: 19689323
references: Biophys J. 2009 Jul 22;97(2):678-86
references: 19619483
references: Chemphyschem. 2007 Feb 19;8(3):433-43
references: 17269116
references: Curr Pharm Biotechnol. 2004 Apr;5(2):155-61
references: 15078149
references: Biophys J. 2005 Aug;89(2):1317-27
references: 15908582
references: Biophys J. 2000 Aug;79(2):1129-38
references: 10920042
references: Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14398-403
references: 11724946
references: Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10909-14
references: 22711840
references: J Microsc. 2000 Oct;200(Pt 1):14-25
references: 11012824
references: J Biomed Opt. 2009 May-Jun;14(3):034047
references: 19566339
references: Biophys J. 2009 Oct 21;97(8):2368-76
references: 19843469
references: Methods Mol Biol. 2014;1076:653-82
references: 24108649
references: Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8936-41
references: 15175430
references: J Cell Sci. 2004 Nov 1;117(Pt 23):5521-34
references: 15479718
references: Anal Chem. 2001 May 1;73(9):2078-86
references: 11354494
references: Biophys J. 2008 May 15;94(10):4103-13
references: 18234806
references: Biopolymers. 1974 Jan;13(1):29-61
references: 4818131
references: Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2170-5
references: 19168634