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Thousands of fluorescent probes are known, and it is not practical to describe them all. This chapter contains an overview of the various types of fluorophores, their spectral properties, and applications. Fluorophores can be broadly divided into two main classes—intrinsic and extrinsic. Intrinsic fluorophores are those that occur naturally. These include the aromatic amino acids, NADH, flavins, derivatives of pyridoxyl, and chlorophyll. Extrinsic fluorophores are added to the sample to provide fluorescence when none exists, or to change the spectral properties of the sample. Extrinsic fluorophores include dansyl, fluorescein, rho-damine, and numerous other substances.

Southwick PL, Ernst LA, Tauriello EW, Parker SR, Mujumdar RB, Mujumdar SW, Clever HA, Waggoner AS. 1990. Cyanine dye labeling reagents-carboxymethylindocyanine succinimidyl esters. Cytometry 11:418–430.

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Vaccari S, Benci S, Peracchi A, Mozzarelli A. 1996. Time-resolved fluorescence of tryptophan synthase. Biophys Chem 61:9–22.

Kao WY, Davis CE, Kim YI, Beach JM. 2001. Fluorescence emission spectral shift measurements of membrane potential in single cells. Biophys J 81:1163–1170.

Balzani V, Ballardini R. 1990. New trends in the design of luminescent metal complexes. Photochem Photobiol 52(2):409–416.

Lin Y, Weissleder R, Tung CH. 2003. Synthesis and properties of sulfhydryl-reactive near-infrared cyanine fluorochromes for fluorescence imaging. Mol Imaging 2(2):87–92.

Karasawa S, Araki T, Yamamoto-Hino M, Miyawaki A. 2003. A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J Biol Chem 278(36):34167–34171.

Loontiens FG, McLaughlin LW, Diekmann S, Clegg RM. 1991. Binding of Hoechst 33258 and 4′,6-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substitutents. Biochemistry 30:182–189.

Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA. 1999. Fluorescent proteins from non-bioluminescent Anthozoa species. Nature Biotechnol 17:969–973.

Loura LMS, Prieto M. 1997. Aggregation state of dehydroergosterol in water and in a model system of membranes. J Fluoresc 7(1):173S–175S.

Thompson RB. 1994. Red and near-infrared fluorometry. In Topics in fluorescence spectroscopy, Vol 4: Probe design and chemical sensing, pp. 151–152. Ed JR Lakowicz. Plenum Press, New York.

Mataga N, Chosrowjan H, Taniguchi S, Tanaka F, Kido N, Kitamura M. 2002. Femtosecond fluorescence dynamcis of flavoproteins: comparative studies on flavodoxin, its site-directed mutants, and riboflavin binding protein regarding ultrafast electron transfer in protein nanospaces. J Phys Chem B 106:8917–8920.

Bowen CM, Katzenellenbogen JA. 1997. Synthesis and spectroscop-ic characterization of two aza-tetrahydrochrysenes as potential fluorescent ligands for the estrogen receptor. J Org Chem 62:7650–7657.

Madhuri S, Vengadesan N, Aruna P, Koteeswaran D, Venkatesan P, Ganesan S. 2003. Native fluorescence spectroscopy of blood plasma in the characterization of oral malignancy. Photochem Photobiol 78(2):197–204.

Vaccari S, Benci S, Peracchi A, Mozzarelli A. 1997. Time-resolved fluorescence of pyridoxal 5′-phosphate-containing enzymes: trypto-phan synthetase and O-acetylserine sulfhydrylase. J Fluoresc 7(1):135S–137S.

Griffin BA, Adams SR, Tsien RY. 1998. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269–272.

Slavik J. 1982. Anilinonaphthalene sulfonate as a probe of membrane composition and function. Biochim Biophys Acta 694:1–25.

Kung CE, Reed JK. 1986. Microviscosity measurements of phospho-lipid bilayers using fluorescent dyes that undergo torsional relaxation. Biochemistry 25:6114–6121. See also Biochemistry (1989) 28:6678–6686.

Li M, Selvin PR. 1995. Luminescent polyaminocarboxylate chelates of terbium and europium: the effect of chelate structure. J Am Chem Soc 117:8132–8138.

Hammer RP, Owens CV, Hwang SH, Sayes CM, Soper SA. 2002. Asymmetrical, water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides. Bioconjugate Chem 13:1244–1252.

Nakanishi J, Nakajima T, Sato M, Ozawa T, Tohda K, Umezawa Y. 2001. Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells. Anal Chem 73:2920–2928.

Matayoshi ED, Wang GT, Krafft GA, Erickson J. 1990. Novel fluo-rogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247:954–957.

Lippincott-Schwartz J, Patterson GH. 2003. Development and use of fluorescent protein markers in living cells. Science 300:87–91.

Zhang J, Davidson RM, Wei M, Loew LM. 1998. Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons. Biophys J 74:48–53.

Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP, Markelov ML, Zaraisky AG, Zhao X, Fang Y, Tan W, Lukyanov SA. 2000. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem 275(34):25879–25882.

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Chang PY, Jackson MB. 2003. Interpretation and optimization of absorbance and fluorescence signals from voltage-sensitive dyes. J Membr Biol 196:105–116.

Klonis N, Wang H, Quazi NH, Casey JL, Neumann GM, Hewish DR, Hughes AB, Deady LW, Tilley L. 2001. Characterization of a series of far red absorbing perylene diones: a new class of fluorescent probes for biological applications. J Fluoresc 11(1):1–11.

Fluorophore structure

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Hemmila I. 1993. Progress in delayed fluorescence immunoassay. In Fluorescence spectroscopy, new methods and applications, pp. 259–266. Ed OS Wolfbeis. Springer-Verlag, New York.

How dofluorophoreswork

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Li L, Szmacinski H, Lakowicz JR. 1997. Long-lifetime lipid probe containing a luminescent metal-ligand complex. Biospectroscopy 3:155–159.

Lamture JB, Wensel TG. 1995. Intensely luminescent immunoreac-tive conjugates of proteins and dipicolinate-based polymeric Tb(III) chelates. Bioconjugate Chem 6:88–92.

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Kwon O-S, Blazquez M, Churchich JE. 1994. Luminescence spec-troscopy of pyridoxic acid and pyridoxic acid bound to proteins. Eur J Biochem 219:807–812.

Fluorophoresexamples

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Billinton N, Knight AW. 2001. Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. Anal Biochem 291:175–197.

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Palmer GM, Keely PJ, Breslin TM, Ramanujam N. 2003. Autfluorescence spectroscopy of normal and malignant human breast cell lines. Photochem Photobiol 78(5):462–469.

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Fluorochrome vs fluorophore

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Is GFP a fluorophore

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Biologicalfluorophores

116 mm | Box of 1000 | 4.5 x 0.75-in

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Fluorophoreslist

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Loos D, Cotlet M, De Schryver F, Habuchi S, Jofkens J. 2004. Single-molecule spectroscopy selectively probes donor and acceptor chromophore in the phycobiliprotein allophycocyanin. Biophys J 87:2598–2608.

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Adams SR, Campbell RE, Gross LA, Martin BR, Walkup GK, Yao Y, Llopis J, Tsien RY. 2002. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 124:6063–6076.

Cacciatore TW, Brodfuehrer PD, Gonzalez JE, Jiang T, Adams SR, Tsien RY, Kristan Jr WB, Kleinfield D. 1999. Identification of neural circuits by imaging coherent electrical activity with FRET-based dyes. Neuron 23:449–459.

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Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung C-Y, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP. 2003. Quantitative comparison of long-wavelength Alexa fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51(12):1699–1712.

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Fluorescence probes represent the most important area of fluorescence spectroscopy. The wavelength and time resolution required of the instruments is determined by the spectral properties of the fluorophores. Furthermore, the information available from the experiments is determined by the properties of the probes. Only probes with non-zero anisotropies can be used to measure rotational diffusion, and the lifetime of the fluorophore must be comparable to the timescale of interest in the experiment. Only probes that are sensitive to pH can be used to measure pH. And only probes with reasonably long excitation and emission wavelengths can be used in tissues, which display autofluorescence at short excitation wavelengths.