The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented.

J Heyrovský Institute of Physical Chemistry v v i Academy of Sciences of the Czech Republic Dolejškova 3 Prague 18223 Czech Republic

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Akyuz M, Cabuk H. Particle-associated polycyclic aromatic hydrocarbons in the atmospheric environment of Zonguldak. Turkey Sci Total Environ. 2008;405:62–70. PubMed

Hashi Y, Wang TR, Du W, Lin JM. Rapid and sensitive determination of polycyclic aromatic hydrocarbons in atmospheric particulates using fast high-performance liquid chromatography with on-line enrichment system. Talanta. 2008;74:986–991. PubMed

Toriba A, Kuramae Y, Chetiyanukornkul T, Kizu R, Makino T, Nakazawa H, Hayakawa K. Quantification of polycyclic aromatic hydrocarbons (PAHs) in human hair by HPLC with fluorescence detection: a biological monitoring method to evaluate the exposure to PAHs. Biomed Chromatogr. 2003;17:126–132. PubMed

Bortolato SA, Arancibia JA, Escandar GM. Non-trilinear chromatographic time retention-fluorescence emission data coupled to chemometric algorithms for the simultaneous determination of 10 polycyclic aromatic hydrocarbons in the presence of interferences. Anal Chem. 2009;81:8074–8084. PubMed

Rodriguez-Acuna R, Perez-Camino Mdel C, Cert A, Moreda W. Sources of contamination by polycyclic aromatic hydrocarbons in Spanish virgin olive oils. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008;25:115–122. PubMed

Katoh T, Yokoyama S, Sanada Y. Analysis of a coal-derived liquid using highpressure liquid chromatography and synchronous fluorescence spectrometry. Fuel. 1980;59:845–850.

Tasis D, Mikroyannidis J, Karoutsos V, Galiotis C, Papagelis K. Single-walled carbon nanotubes decorated with a pyrene-fluorenevinylene conjugate. Nanotechnology. 2009;20:135606. PubMed

Williams WB, Mullany BA, Parker WC, Moyer PJ, Randles MH. Using quantum dots to tag subsurface damage in lapped and polished glass samples. Appl Opt. 2009;48:5155–5163. PubMed

Bhatta H, Goldys EM, Learmonth RP. Use of fluorescence spectroscopy to differentiate yeast and bacterial cells. Appl Microbiol Biotechnol. 2006;71:121–126. PubMed

Ross JA, Jameson DM. Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence. Photochem Photobiol Sci. 2008;7:1301–1312. PubMed

James NG, Ross JA, Mason AB, Jameson DM. Excited-state lifetime studies of the three tryptophan residues in the N-lobe of human serum transferrin. Protein Sci. 2009;19:99–110. PubMed PMC

Parasassi T, Conti F, Gratton E, Sapora O. Membranes modification of differentiating proerythroblasts. Variation of 1,6-diphenyl-1,3,5-hexatriene lifetime distributions by multifrequency phase and modulation fluorimetry. Biochim Biophys Acta. 1987;898:196–201. PubMed

Fiorini R, Valentino M, Wang S, Glaser M, Gratton E. Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles. Biochemistry. 1987;26:3864–3870. PubMed

Clegg RM, Murchie AI, Zechel A, Carlberg C, Diekmann S, Lilley DM. Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry. 1992;31:4846–4856. PubMed

Celli A, Sanchez S, Behne M, Hazlett T, Gratton E, Mauro T. The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging. Biophys J. 2010;98:911–921. PubMed PMC

Weber G. Enumeration of components in complex systems by fluorescence spectrophotometry. Nature. 1961;190:27–29. PubMed

Chen J, Lee A, Zhao J, Wang H, Lui H, McLean DI, Zeng H. Spectroscopic characterization and microscopic imaging of extracted and in situ cutaneous collagen and elastic tissue components under two-photon excitation. Skin Res Technol. 2009;15:418–426. PubMed

Kavanagh RJ, Burnison BK, Frank RA, Solomon KR, Van Der Kraak G. Detecting oil sands process-affected waters in the Alberta oil sands region using synchronous fluorescence spectroscopy. Chemosphere. 2009;76:120–126. PubMed

Valeur B. Molecular Fluorescence. Wiley-VCH; Weiheim, Germany: 2002.

Lakowicz J. Principles of Fluorescence Spectroscopy. Springer; New York: 2006.

Smyk B, Amarowicz R, Szabelski M, Gryczynski I, Gryczynski Z. Steady-state and time-resolved fluorescence studies of stripped Borage oil. Anal Chim Acta. 2009;646:85–89. PubMed

Bright FV, Betts TA, Litwiler KS. Advances in Multifrequency Phase and Modulation Fluorescence Analysis. Crit Rev Anal Chem. 1990;21:389–405.

Weber G. Resolution of the fluorescence lifetimes in a heterogeneous system by phase and modulation measurements. J Phys Chem. 1981;85:949–953.

Barbieri B, Terpetschnig E, Jameson DM. Frequency-domain fluorescence spectroscopy using 280-nm and 300-nm light-emitting diodes: measurement of proteins and protein-related fluorophores. Anal Biochem. 2005;344:298–300. PubMed

Gratton E, Jameson DM, Rosato N, Weber G. Multifrequency cross-correlation phase fluorometer using synchrotron radiation. Rev Sci Instrum. 1984;55:486–494.

Alcala JR, Gratton E, Prendergast FG. Fluorescence lifetime distributions in proteins. Biophys J. 1987;51:597–604. PubMed PMC

Brochon JC. Maximum entropy method of data analysis in time-resolved spectroscopy. Methods Enzymol. 1994;240:262–311. PubMed

Jameson DM, Gratton E, Hall RD. The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry. Appl Spectrosc Rev. 1984;20:55–106. PubMed

Reinhart GD, Marzola P, Jameson DM, Gratton E. A method for on-line background subtraction in frequency domain fluorometry. J Fluoresc. 1991;1:153–162. PubMed

Clayton AH, Hanley QS, Arndt-Jovin DJ, Subramaniam V, Jovin TM. Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM) Biophys J. 2002;83:1631–1649. PubMed PMC

Clayton AHA, Hanley QS, Verveer PJ. Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data. J Microsc. 2004;213:1–5. PubMed

Redford GI, Clegg RM. Polar plot representation for frequency-domain analysis of fluorescence lifetimes. J Fluoresc. 2005;15:805–815. PubMed

Hanley QS, Clayton AHA. AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers. J Microsc. 2005;218:62–67. PubMed

Esposito A, Gerritsen HC, Oggier T, Lustenberger F, Wouters FS. Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics. J Biomed Opt. 2006;11:34016. PubMed

Digman MA, Caiolfa VR, Zamai M, Gratton E. The phasor approach to fluorescence lifetime imaging analysis. Biophys J. 2008;94:L14–L16. PubMed PMC

Clayton AHA. The polarized AB plot for the frequency-domain analysis and representation of fluorophore rotation and resonance energy homotransfer. J Microsc. 2008;232:306–312. PubMed

Stringari C, Digman M, Donovan P, Gratton E. Multiple Components Mapping of Live Tissue by Phasor Analysis of Fluorescence Lifetime Imaging. Biophys J. 2010;98:214a–214a.

Redford GI, Majumdar ZK, Sutin JD, Clegg RM. Properties of microfluidic turbulent mixing revealed by fluorescence lifetime imaging. J Chem Phys. 2005;123:224504. PubMed

Teale FWJ. Phase and Modulation Fluorometry. In: Cundall RB, Dale RE, editors. Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology. Plenum Press; New York: 1983. pp. 59–80.

Weber G. Polarization of the fluorescence of macromolecules. II. Fluorescent conjugates of ovalbumin and bovine serum albumin. Biochem J. 1952;51:155–167. PubMed PMC

Parasassi T, Conti F, Gratton E. Time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry. Cell Mol Biol. 1986;32:103–108. PubMed

Spencer RD, Weber G. Influence of Brownian Rotations and Energy Transfer upon the Measurements of Fluorescence Lifetime. J Chem Phys. 1970;52:1654–1663.

Bismuto E, Irace G, Colonna G, Jameson DM, Gratton E. Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins. Biochim Biophys Acta. 1987;913:150–154. PubMed

Gratton E, Limkeman M. A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys J. 1983;44:315–324. PubMed PMC

Jameson DM, Gratton E. Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry. In: Eastwood D, editor. New Directions In Molecular Luminescence. American Society for Testing and Materials; Philadelphia: 1983. pp. 67–81.

Bismuto E, Jameson DM, Gratton E. Dipolar relaxations in glycerol: a dynamic fluorescence study of 4-[2′-(dimethylamino)-6′-naphthoyl]cyclohexanecarboxylic acid (DANCA) J Am Chem Soc. 1987;109:2354–2357.

Weber G, Farris FJ. Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry. 1979;18:3075–3078. PubMed

Caffrey M, Hogan J. LIPIDAT: a database of lipid phase transition temperatures and enthalpy changes. DMPC data subset analysis. Chem Phys Lipids. 1992;61:1–109. PubMed

Parasassi T, Ravagnan G, Rusch RM, Gratton E. Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence. Photochem Photobiol. 1993;57:403–410. PubMed

Parasassi T, Di Stefano M, Loiero M, Ravagnan G, Gratton E. Influence of cholesterol on phospholipid bilayers phase domains as detected by Laurdan fluorescence. Biophys J. 1994;66:120–132. PubMed PMC

Merlo S, Yager P, Burgess W. An optical method for detecting anestheics and other lipid soluble compounds. Sensors and Actuators. 1990;A23:1150–1154.

Weber G, Young LB. Fragmentation of Bovine Serum Albumin by Pepsin. I. The Origin of the Acid Expansion of the Albumin Molecule. J Biol Chem. 1964;239:1415–1423. PubMed

Stryer L. The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol. 1965;13:482–495. PubMed

Gafni A, DeToma RP, Manrow RE, Brand L. Nanosecond decay studies of a fluorescence probe bound to apomyoglobin. Biophys J. 1977;17:155–168. PubMed PMC

Cheung HC, Gryczynski I, Malak H, Wiczk W, Johnson ML, Lakowicz JR. Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry. Biophysical Chemisrty. 1991;40:1–17. PubMed

Shih WM, Gryczynski Z, Lakowicz JR, Spudich JA. A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell. 2000;102:683–694. PubMed

Chen YC, Spring BQ, Buranachi C, Tong B, Malachowski G, Clegg RM. General Concerns of FLIM Data Representation and Analysis. In: Perisasamy A, Clegg RM, editors. FLIM Microscopy in Biology and Medicine. Chapman & Hall/CRC Press; Boca Raton: 2010.

Chen YC, Clegg RM. Fluorescence lifetime-resolved imaging. Photosynth Res. 2009 PubMed

Visser NV, Borst JW, Hink MA, van Hoek A, Visser AJ. Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophys Chem. 2005;116:207–212. PubMed

PMP22 duplication dysregulates lipid homeostasis and plasma membrane organization in developing human Schwann cells