Visser AJWG. 1984. Kinetics of stacking interactions in flavin adenine dinucleotide from time-resolved flavin fluorescence. Photochem Photobiol 40(6):703–706.

Baird GS, Zacharias DA, Tsien RY. 2000. Biochemistry, mutagene-sis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97(22):11984–11989.

Murphys JT, Lagarias JC. 1997. Purification and characterization of recombinant affinity peptide-tagged oat phytochrome A. Photochem Photobiol 65(4):750–758.

Bruno J, Horrocks WDeW, Zauhar RJ. 1992. Europium(III) luminescence and tyrosine to terbium(III) energy transfer studies of invertebrate (octopus) calmodulin. Biochemistry 31:7016–7026.

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.

Prendergast FG, Haugland RP, Callahan PJ. 1981. 1-[4-(trimethy-lamino)phenyl]-6-phenylhexa-1,3,5 triene: synthesis, fluorescence properties, and use as a fluorescence probe of lipid bilayers. Biochemistry 20:7333–7338.

Leenders R, Kooijman M, van Hoek A, Veeger C, Visser AJWG. 1993. Flavin dynamics in reduced flavodoxins. Eur J Biochem 211:37–45.

Prendergast FG, Meyer M, Carlson GL, Iida S, Potter JD. 1983. Synthesis, spectral properties, and use of 6-acryloyl-2-dimethy-laminonaphthalene (Acrylodan). J Biol Chem 258(12):7541–7544.

Oi VT, Glazer AN, Stryer L. 1982. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J Cell Biol 93:981–986.

Figure 3: Gaussian beam parameters such as the radius, W(z), and the wavefront's radius of curvature, R(z), are calculated using a distance, z, referenced to the beam's waist. The waist is always located at the origin.

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.

Andresen M, Schmitz-Salue R, Jakobs S. 2004. Short tetracysteine tags to β-tubulin demonstrate the significance of small labels for live cell imaging. Mol Biol Cell 15:5616–5622.

Terpetschnig E, Szmacinski H, Lakowicz JR. 1997. Long lifetime metal—ligand complexes as probes in biophysics and clinical chemistry. Methods Enzymol 278:295–321.

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

Valeur B. 1994. Principles of fluorescent probe design for ion recognition. In Topics in fluorescence spectroscopy, Vol. 4: Probe design and chemical sensing, pp. 21–48. Ed JR Lakowicz. Plenum Press, New York.

Gershkovich AA, Kholodovych VV. 1996. Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods 33:135–162.

Hawkins ME, Pfleiderer W, Mazumder A, Pommier YG, Balis FM. 1995. Incorporation of a fluorescent guanosine analog into oligonu-cleotides and its application to a real time assay for the HIV-1 integrase 3′-processing reaction. Nucleic Acids Res 23(15):2872–2880.

Jean JM, Hall KB. 2001. 2-aminopurine fluorescence quenching and lifetimes: role of base stacking. Proc Natl Acad Sci USA 98(1):37–41.

Rettig W, Lapouyade R. 1994. Fluorescence probes based on twisted intramolecular charge transfer (TICT) states and other adiabatic pho-toreactions. In Topics in fluorescence spectroscopy, Vol. 4: Probe design and chemical sensing, pp. 109–149. Ed JR Lakowicz. Plenum Press, New York.

Buschmann V, Weston KD, Sauer M. 2003. Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem 14:195–204.

Xiao G-S, Zhou J-M. 1996. Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5′-phosphate. Biochim Biophys Acta 1294:1–7.

Schroeder F, Barenholz Y, Gratton E, Thompson TE. 1987. A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles. Biochemistry 26:2441–2448.

Sabbatini N, Guardigli M. 1993. Luminescent lanthanide complexes as photochemical supramolecular devices. Coord Chem Rev 123:201–228.

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.

Note that the beam radius (W ) and wavefront's radius of curvature (R ) are calculated using the relative distance (z ) from the waist (Figure 3). There are two modifed thin-lens equations (table, bottom), since the form of the equation differs depending on whether the input beam parameters (zR and s ) or the output beam parameters (zR ' and s ') are known.

If the laser beam is not perfectly Gaussian, its Rayleigh range will be shorter than the Rayleigh range of an ideal Gaussian beam with the same waist radius. The ratio of the two values, M2 (M-squared), is often specified for laser beams. Multiply the beam's Rayleigh range by the M2 value, and then use this product as zR or zR ' in the modified thin-lens equation to find s ' or s , respectively.

Objects, and Images, and Laser BeamsThe conventional thin-lens equation (table, top) is based on the ray optics model and uses the focal length (f ) of the lens to relate the distance (s ) between the lens and the object to the distance (s ') between the lens and the image.

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802–805.

Walkup GK, Imperiali B. 1996. Design and evaluation of a peptidyl fluorescent chemosensor for divalent zinc. J Am Chem Soc 118:3053–3054.

Geddes CD, Lakowicz JR, eds. 2005. Advanced concepts in fluorescence sensing: small molecule sensing. Top Fluoresc Spectrosc 9, forthcoming.

Holzwarth AR, Wendler J, Suter GW. 1987. Studies on chromophore coupling in isolated phycobiliproteins. Biophys J 51:1–12.

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.

Douglass PM, Salins LLE, Dikici E, Daunert S. 2002. Class-selective drug detection: fluorescently labeled calmodulin as the biorecogni-tion element for phenothiazines and tricyclic antidepressants. Bioconjugate Chem 13:1186–1192.

Demas JN, DeGraff BA. 1992. Applications of highly luminescent transition metal complexes in polymer systems. Macromol Chem Macromol Symp 59:35–51.

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.

Telford WG, Moss MW, Morseman JP, Allnutt FCT. 2001. Cyanobacterial stabilized phycobilisomes as fluorochromes for extracellular antigen detection by flow cytometry. J Immunol Methods 254:13–30.

DaCosta RS, Andersson H, Wilson BC. 2003. Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy. Photochem Photobiol 78(4):384–392.

Modified Thin-Lens EquationThe incident and output light are assumed to propagate as Gaussian beams (Figure 2). The modified thin-lens equation was derived by relating the input and output beam properties (table, center) across the lens:

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.

Bevis BJ, Glick BS. 2002. Rapidly maturing variants of the discosoma red fluorescent protein (DsRed). Nature Biol 20:83–87.

Nakanishi J, Maeda M, Umezawa Y. 2004. A new protein conformation indicator based on biarsenical fluorescein with an extended ben-zoic acid moiety. Anal Sci 20:273–278.

Clarke RJ, Kane DJ. 1997. Optical detection of membrane dipole potential: avoidance of fluidity and dye-induced effects. Biochim Biophys Acta 1323:223–239.

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

ABCD matrixGaussianbeam

Wu P, Li H, Nordlund TM, Rigler R. 1990. Multistate modeling of the time and temperature dependence of fluorescence from 2-aminopurine in a DNA decamer. SPIE Proc 204:262–269.

Brochon J-C, Wahl P, Monneuse-Doublet M-O, Olomucki A. 1977. Pulse fluorimetry study of octopine dehydrogenase-reduced nicoti-namide adenine dinucleotide complexes. Biochemistry 16(21):4594–4599.

Gonzalez JE, Tsien RY. 1995. Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys J 69:1272–1280.

Triantafilou K, Triantafilou M, Wilson KM. 2000. Phycobili-protein–fab conjugates as probes for single particle fluorescence imaging. Cytometry 41:226–234.

Ignatova Z, Gierasch LM. 2004. Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc Natl Acad Sci USA 101:523–528.

Ehrig T, O’Kane DJ, Prendergast FG. 1995. Green fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett 367:163–166.

Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395.

Kao JPY. 1994. Practical aspects of measuring [Ca2+] with fluorescent indicators. In Methods in Cell Biology, Vol. 40, pp. 155–181. Ed R Nuccitelli. Academic Press, New York.

Plásek J, Sigler K. 1996. Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis. J Photochem Photobiol B: Biol 33:101–124.

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.

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

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.

Sims PJ, Waggoner AS, Wang C-H, Hoffman JF. 1974. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13(16):3315–3336.

Glazer AN, Peck K, Matheis RA. 1990. A stable double-stranded DNA ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels. Proc Natl Acad Sci USA 87:3851–3855.

Gaussianbeam intensity formula

Gafni A, Brand L. 1976. Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase. Biochemistry 15(15):3165–3171.

Lumture JB, Wensel TG. 1993. A novel reagent for labelling macro-molecules with intensity luminescent lanthanide complexes. Tetrahedron Lett 34(26):4141–4144.

Flanagan JH, Romero SE, Legendre BL, Hammer RP, Soper A. 1997. Heavy-atom modified near-IR fluorescent dyes for DNA sequencing applications: synthesis and photophysical characterization. SPIE Proc 2980:328–337.

Churchich JE. 1976. Fluorescent probe studies of binding sites in proteins and enzymes. Mod Fluoresc Spectrosc 2:217–237.

Johnson ID, Kang HC, Haugland RP. 1991. Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores. Anal Biochem 198:228–237.

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

Sklar LA, Hudson BS, Petersen M, Diamond J. 1977. Conjugated polyene fatty acids on fluorescent probes: Spectroscopic characterization. Biochemistry 16(5):813–818.

Gambetta GA, Lagarias JC. 2001. Genetic engineering of phy-tochrome biosynthesis in bacteria. Proc Natl Acad Sci USA 98(19):10566–10571.

Churchich JE. 1986. Fluorescence properties of free and bound pyri-doxal phosphate and derivatives. Pyridoxal Phosphate: Chem Biochem Med Asp A, 1A:545–567.

Daniel E, Weber G. 1966. Cooperative effects in binding by bovine serum albumin, I: the binding of 1-anilino-8-naphthalenesulfonate. Fluorimetric titrations. Coop Effects Binding Albumin 5:1893–1900.

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.

Lövgren T, Pettersson K. 1990. Time-resolved fluoroimmunoassay, advantages and limitations. In Luminescence immunoassay and molecular applications, pp. 233–253. Ed K Van Dyke, R Van Dyke. CRC Press, Boca Raton, FL.

Arden-Jacob J, Frantzeskos J, Kemnitzer NU, Zilles A, Drexhage KH. 2001. New fluorescent markers for the red region. Spectrochim Acta A 57:2271–2283.

Gaussianbeam waist

Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY. 2002. A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99(12):7877–7882.

Oswald B, Gruber M, Bohmer M, Lehmann F, Probst M, Wolfbeis OS. 2001. Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay. Photochem Photobiol 74(2):237–245.

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.

Tjioe I, Legerton T, Wegstein J, Herzenberg LA, Roederer M. 2001. Phycoerythrin-allophycocyanin: a resonance energy transfer fluo-rochrome for immunofluorescence. Cytometry 44:24–29.

Kinnunen PKJ, Koiv A, Mustonen P. 1993. Pyrene-labeled lipids as fluorescent probes in studies on biomembranes and membrane models. In Fluorescence spectroscopy: new methods and applications, pp. 159–171. Ed OS Wolfbeis. Springer-Verlag, New York.

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.

Albani JR, Sillen A, Engelborghs Y, Gervais M. 1999. Dynamics of flavin in flavocytochrome b2: a fluorescence study. Photochem Photobiol 69(1):22–26.

Figure 2: Key to the model are the relationships between the input and output beams at the lens. There, the beams have equal radii, and the beams' wavefront curvatures are related by the focal length of the lens. The wavefront' radius of curvature is flat (thick vertical line) at the waist and gradually becomes more spherical with increasing distance from the waist.

Rye HS, Yue S, Wemmer DE, Quesada MA, Haugland RP, Mathies RA, Glazer AN. 1992. Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications. Nucleic Acids Res 20(11):2803–2812.

Zimmer M. 2002. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759–781.

Richards-Kortum R, Sevick-Muraca E. 1996. Quantitative optical spectroscopy for tissue diagnosis. Annu Rev Phys Chem 47:555–606.

Gross E, Bedlack RS, Loew LM. 1994. Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential. Biophys J 67:208–216.

Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY. 1995. Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455.

Geoghegan KF. 1996. Improved method for converting an unmodified peptide to an energy-transfer substrate for a proteinase. Bioconjugate Chem 7(3):385–391.

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.

Rahavendran SV, Karnes HT. 1996. An oxazine reagent for derivati-zation of carboxylic acid analytes suitable for liquid chromatograph-ic detection using visible diode laser-induced fluorescence. J Pharmacol Biomed Anal 15:83–98.

Czarnik AW. 1994. Fluorescent chemosensors for cations, anions, and neutral analytes. In Topics in fluorescence spectroscopy, Vol. 4: probe design and chemical sensing, pp. 49–70. 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.

Gaussian laserbefore and after

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.

In the modified thin-lens equation, these distances refer to the separations between the lens and the input and output beam waists, respectively (Figure 1). The radius of a beam is smallest at its waist and increases with distance. A lens placed in the beam outputs a new beam with its own waist and divergence characteristics. The input beam waist radius (Wo ) is treated as the object size, and output beam waist radius (Wo' ) as the image size.

Breton R, Housset D, Mazza C, Fontecilla-Camps JC. 1996. The structure of a complex of human 17-β-hydroxysteroid dehydroge-nase with estradiol and NADP+ identifies two principal targets for the design of inhibitors. Structure 4:905–915.

Richardson FS. 1982. Terbium(III) and europium(III) ions as luminescent probes and stains for biomolecular systems. Chem Rev 82:541–552.

Szmacinski H, Lakowicz JR. 1994. Lifetime-based sensing. In Topics in fluorescence spectroscopy, Vol. 4: Probe design and chemical sensing, pp. 295–334. Ed JR Lakowicz. Plenum Press, New York.

Davenport L, Targowski P. 1996. Submicrosecond phospholipid dynamics using a long lived fluorescence emission anisotropy probe. Biophys J 71:1837–1852.

Li B, Lin S-X. 1996. Fluorescence-energy transfer in human estradi-ol 17β-dehydrogenase–NADH complex and studies on the coenzyme binding. Eur J Biochem 235:180–186.

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

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.

Hwang K-J, O’Neil JP, Katzenellenbogen JA. 1992. 5,6,11,12-Tetrahydrochrysenes: synthesis of rigid stilbene systems designed to be fluorescent ligands for the estrogen receptor. J Org Chem 57:1262–1271.

Graefe KA, Tang Z, Karnes HT. 2000. High-performance liquid chromatography with on-line post-column immunoreaction detection of digoxin and its metabolites based on fluorescence energy transfer in the far-red spectral region. J Chromatogr B 745:305–314.

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.

Telford WG, Moss MW, Morseman JP, Allnutt FCT. 2001. Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry. Cytometry 44:16–23.

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.

Gaussianbeam pdf

In the case of laser light, a modified thin-lens equation that takes diffraction into account is recommended instead of the conventional thin-lens equation. The modified equation, which models the laser light as Gaussian beams, is appropriate for many single-mode and fiber-coupled laser sources. In addtion, the equation can be adapted for use when the laser light does not have a perfect Gaussian intensity profile. [1]

Hull RV, Conger PS, Hoobler RJ. 2001. Conformation of NADH studied by fluorescence excitation transfer spectroscopy. Biophys Chem 90:9–16.

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.

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.

Zhou M, Upson RH, Diwu Z, Haugland RP. 1996. A fluorogenic substrate for β-glucuronidase: applications in fluorometric, polyacry-lamide gel and histochemical assays. J Biochem Biophys Methods 33:197–205.

Martin RB, Richardson FS. 1979. Lanthanides as probes for calcium in biological systems. Quart Rev Biophys 12(2):181–209.

Rahavendran SV, Karnes HT. 1996. Application of rhodamine 800 for reversed phase liquid chromatographic detection using visible diode laser induced fluorescence. Anal Chem 68:3763–3768.

Wolfbeis OS. 1985. The fluorescence of organic natural products. In Molecular luminescence spectroscopy, Part 1, pp. 167–370. Ed SG Schulman. John Wiley & Sons, New York.

Figure 1: A thin lens, with focal length f , is shown inserted in a Gaussian beam. In the modified thin-lens equation, the object is the input beam's waist, located a distance s  from the input side of the lens. The input beam's radius (W ) is Wo at its waist and maintains a similar radius over the Rayleigh range (±zR ). The image is the output beam's waist, located a distance s ' from the lens' output side. The output beam's radius (W ') is Wo' at its waist and remains nearly Wo ' over its Rayleigh range (±zR').

Gaussianbeam equation

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.

Nordlund TM, Wu P, Anderson S, Nilsson L, Rigler R, Graslund A, McLaughlin LW, Gildea B. 1990. Structural dynamics of DNA sensed by fluorescence from chemically modified bases. SPIE Proc 1204:344–353.

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

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.

Eriksson S, Kim SK, Kubista M, Norden B. 1993. Binding of 4′,6-diamidino-2-phenylindole (DAPI) to AT regions of DNA: evidence for an allosteric conformational change. Biochemistry 32:2987–2998.

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.

Klymchenko AS, Duportail G, Mély Y, Demchenko AP. 2003. Ultrasensitive two-color fluorescence probes for dipole potential in phospholipid membranes. Proc Natl Acad Sci USA 100(20):11219–11224.

Trinquet E, Maurin F, Préaudat M, Mathis G. 2001. Allphycocyanin 1 as a near-infrared fluorescent tracer: isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system. Anal Biochem 296:232–244.

Li L, Murphy JT, Lagarias JC. 1995. Continuous fluorescence assay of phytochrome assembly in vitro. Biochemistry 34:7923–7930.

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.

Shapovalov VL, Kotova EA, Rokitskaya TI, Antonenko YN. 1999. Effect of Gramicidin A on the dipole potential of phospholipid membranes. Biophys J 77:299–305.

Longworth JW. 1971. Luminescence of polypeptides and proteins. In Excited states of proteins and nucleic acids, pp. 319–484. Ed RF Steiner, I Welnryb, Plenum, New York.

Adachi M, Nagao Y. 2001. Design of near-infrared dyes based on A-conjugation system extension, 2: theoretical elucidation of framework extended derivatives of perylene chromophore. Chem Mater 13:662–669.

Fischer AJ, Lagarias JC. 2004. Harnessing phytochrome’s glowing potential. Proc Natl Acad Sci USA (Early Ed.) 101:17334–17339.

Wiedenmann J, Schenk A, Rocker C, Girod A, Spindler KD. 2002. A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (anthozoa, actinaria). Proc Natl Acad Sci USA 99(18):11646–11651.

Benson RC, Meyer RA, Zaruba ME, McKhann GM. 1979. Cellular autofluorescence: is it due to flavins? J Histochem Cytochem 27(1):44–48.

Heim R, Tsien RY. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182.

Davenport L, Shen B, Joseph TW, Straher MP. 2001. A novel fluorescent coronenyl-phospholipid analogue for investigations of sub-microsecond lipid fluctuations. Chem Phys Lipids 109:145–156.

For example, the conventional thin-lens equation (see table), predicts the image will appear at infinity when the object is placed at the lens' front focal point. However, for Gaussian beams, when the object beam waist is one focal length away from the input side of the lens, the image beam waist is one focal length away from the output side.

Adir N, Lerner N. 2003. The crystal structure of a novel unmethylat-ed form of C-phycocyanin, a possible connector between cores and rods in phycobilisomes. J Biol Chem 278(28):25926–25932.

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.

Illsley NP, Verkman AS. 1987. Membrane chloride transport measured using a chloride-sensitive fluorescent probe. Biochemistry 26:1215–1219.

Kwon MS, Koo BC, Choi BR, Lee HT, Kim YH, Ryu WS, Shim H, Kim JH, Kim NH, Kim T. 2004. Development of transgenic chickens expressing enhanced green fluorescent protein. Biochem Biophys Res Commun 320:442–448.

Gaussianbeam profile

Unique Behavior of Gaussian BeamsThe behavior of Gaussian beams can be surprising, especially when compared with the predictions of the ray optics model and the conventional thin-lens equation.

Jean JM, Hall KB. 2002. 2-aminopurine electronic structure and fluorescence properties in DNA. Biochemistry 41:13152–13161.

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.

Yang M, Baranov E, Jiang P, Sun FX, Li XM, Li L, Hasegawa S, Bouvet M, Al-tuwaijri M, Chishima T, Shimada H, Moossa AR, Penman S, Hoffman RM. 2000. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci USA 97(3):1206–1211.

Velick SF. 1958. Fluorescence spectra and polarization of glyceralde-hyde-3-phosphate and lactic dehydrogenase coenzyme complexes. J Biol Chem 233:1455–1467.

Wang Q, Scheigetz J, Gilbert M, Snider J, Ramachandran C. 1999. Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases. Biochim Biophys Acta 1431:14–23.

Wagnieres GA, Star WM, Wilson BC. 1998. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem Photobiol 68(5):603–632.

Gaussianbeam calculator

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.

depends on the Rayleigh range of the input beam. For the image beam waist to appear at this distance, the object beam waist should be separated from the lens by a distance one Rayleigh range larger than the focal length, ().

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

Fischer RT, Cowlen MS, Dempsey ME, Schroeder F. 1985. Fluorescence of Δ5,7,9(11),22-ergostatetraen-3β-ol in micelles, sterol carrier protein complexes, and plasma membranes. Biochemistry 24:3322–3331.

Parkinson JA, Barber J, Douglas KT, Rosamond J, Sharples D. 1990. Minor-groove recognition of the self-complementary duplex d(CGC-GAATTCGCG)2 by Hoechst 33258: a high-field NMR study. Biochemistry 29:10181–10190.

Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, Kubota M, Ohashi M, Tsuji FI. 1996. Chemical nature of the light emitter of the Aequorea green fluorescent protein. Proc Natl Acad Sci USA 93:13617–13622.

Steiner RF, Kubota Y. 1983. Fluorescent dye-nucleic acid complexes. In Excited states of biopolymers. Ed RF Steiner. Plenum Press, New York.

Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith TW, Steele GD, Chen LB. 1991. Intracellular heterogeneity in mito-chondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88:3671–3675.

Kawai M, Lee MJ, Evans KO, Nordlund TM. 2001. Temperature and base sequence dependence of 2-aminopurine fluorescence bands in single- and double-stranded oligodeoxynucleotides. J Fluoresc 11(1):23–32.

Poenie M, Chen C-S. 1993. New fluorescent probes for cell biology. In Optical microscopy, pp. 1–25. Ed B Herman, JJ Lemasters. Academic Press, New York.

Fradkov AF, Chen Y, Ding L, Barsova EV, Matz MV, Lukyanov SA. 2000. Novel fluorescent protein from discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett 479:127–130.

Dragsten PR, Webb WW. 1978. Mechanism of the membrane potential sensitivity of the fluorescent membrane probe merocyanine 540. Biochemistry 17:5228–5240.

Mizeret J. 1998. Cancer detection by endoscopic frequency-domain fluorescence lifetime imaging. Thesis presented at École Polytechnique Federale de Lausanne, 177 pp.

In another case, the conventional thin-lens equation predicts the image can be infinitely distant. But, if the beam is Gaussian with a nonzero Rayleigh range, the beam waist cannot be imaged to infinity. Instead, the maximum image beam waist distance,

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

Horrocks WDeW, Sudnick DR. 1981. Lanthanide ion luminescence probes of the structure of biological macromolecules. Acc Chem Res 14:384–392.

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

Hale JE, Schroeder F. 1982. Asymmetric transbilayer distribution of sterol across plasma membranes determined by fluorescence quenching of dehydroergosterol. Eur J Biochem 122:649–661.

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.

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

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

Diegelman S, Fiala A, Leibold C, Spall T, Buchner E. 2002. Transgenic flies expressing the fluorescence calcium sensor cameleon 2.1 under UAS control. Genesis 34:95–98.

(2006). Fluorophores. In: Lakowicz, J.R. (eds) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-46312-4_3

Mizuno H, Sawano A, Eli P, Hama H, Miyawaki A. 2001. Red fluorescent protein from discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry 40:2502–2510.

Huang S, Heikal AA, Webb WW. 2002. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J 82:2811–2825.

The modified thin-lens equation also includes either the input Rayleigh range (zR ) or the output Rayleigh range ( zR '). The Rayleigh range (Rayleigh length) is measured starting at the beam waist, along the propagation direction, to the point where the beam radius is larger by a factor of √2. Highly divergent beams have short Rayleigh ranges.

Haq I, Ladbury JE, Chowdhry BZ, Jenkins TC, Chaires JB. 1997. Specific binding of Hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies. J Mol Biol 271:244–257.

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.