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Fluorescence Spectroscopy: Basic Foundations and Methods

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Analytical Techniques in the Pharmaceutical Sciences

Part of the book series: Advances in Delivery Science and Technology ((ADST))

Abstract

Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundations of the fluorescence phenomenon, introduces some general methodologies and provides selected examples on applications focused to disentangle structural and dynamical aspects of biological processes.

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Notes

  1. 1.

    This compound gives the particular bitter flavor to “tonic water”. Due to the presence of quinine sulfate, this beverage emits blue light when it is illuminated with a UV excitation light source.

  2. 2.

    Absorption cross section is a measure of the probability of light absorption occurring in a given compound.

  3. 3.

    which will be referred to as “fluorophores” in the rest of this chapter (notice that this type of molecules can be also referred as “fluorescent probes”, or “fluorescent dyes”).

  4. 4.

    A transition moment is the electric dipole moment associated with the transition between two electronic states. In general the transition dipole moment is a complex vector quantity. Its direction gives the polarization of the transition, which determines how the system will interact with an electromagnetic wave of a given polarization.

  5. 5.

    The principle of confocal imaging was patented in 1957 by Marvin Minsky and aims to overcome some limitations of traditional wide-field fluorescence microscopes, which lack resolution in the axial direction. A confocal microscope uses scanned point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane a section with variable width (in the order of 0.6 μm or more) can be obtained.

References

  • Acuna AU, Amat-Guerri F, Morcillo P, Liras M, Rodriguez B (2009) Structure and formation of the fluorescent compound of Lignum nephriticum. Org Lett 11(14):3020–3023

    Article  CAS  PubMed  Google Scholar 

  • Alcala JR, Gratton E, Prendergast FG (1987a) Interpretation of fluorescence decays in proteins using continuous lifetime distributions. Biophys J 51(6):925–936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alcala JR, Gratton E, Prendergast FG (1987b) Fluorescence lifetime distributions in proteins. Biophys J 51(4):597–604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alcala JR, Gratton E, Prendergast FG (1987c) Resolvability of fluorescence lifetime distributions using phase fluorometry. Biophys J 51(4):587–596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alvarez-Roman R, Naik A, Kalia YN, Fessi H, Guy RH (2004) Visualization of skin penetration using confocal laser scanning microscopy. Eur J Pharm Biopharm 58(2):301–316

    Article  CAS  PubMed  Google Scholar 

  • Bagatolli LA (2006) To see or not to see: lateral organization of biological membranes and fluorescence microscopy. Biochim Biophys Acta 1758(10):1541–1556

    Article  CAS  PubMed  Google Scholar 

  • Bagatolli LA (2013) LAURDAN fluorescence properties in membranes: a journey from the fluorometer to the microscope. In: Mely Y, Duportail G (eds) Fluorescent methods to study biological membranes, vol 13, Springer series on fluorescence. Springer, Berlin, pp 3–36

    Chapter  Google Scholar 

  • Bagatolli LA, Montich GG, Ravera M, Perez JD, Fidelio GD (1995) Fatty acid indole fluorescent derivatives as probes to measure the polarity of interfaces containing gangliosides. Chem Phys Lipids 78(2):193–202

    Article  CAS  Google Scholar 

  • Bagatolli LA, Kivatinitz SC, Aguilar F, Soto MA, Sotomayor CP, Fidelio GD (1996a) Two distinguishable fluorescent modes of 1-anilino 8-naphthalenesulfonate bound to Human Albumin. J Fluoresc 6:33–40

    Article  CAS  PubMed  Google Scholar 

  • Bagatolli LA, Kivatinitz SC, Fidelio GD (1996b) Interaction of small ligands with human serum albumin IIIA subdomain. How to determine the affinity constant using an easy steady state fluorescent method. J Pharm Sci 85(10):1131–1132

    Article  CAS  PubMed  Google Scholar 

  • Bloksgaard M, Brewer J, Bagatolli LA (2013) Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods. Eur J Pharm Sci 50(5):586–594

    Article  CAS  PubMed  Google Scholar 

  • Bouwstra JA, Honeywell-Nguyen PL (2002) Skin structure and mode of action of vesicles. Adv Drug Deliv Rev 54:S41–S55

    Article  CAS  PubMed  Google Scholar 

  • Boyle R (1664) Experiments and considerations touching colours. Henry Herringman, London

    Google Scholar 

  • Brewer J, Bloksgaard M, Kubiak J, Sorensen JA, Bagatolli LA (2013) Spatially resolved two-color diffusion measurements in human skin applied to transdermal liposome penetration. J Invest Dermatol 133(5):1260–1268

    Article  CAS  PubMed  Google Scholar 

  • Carrer DC, Vermehren C, Bagatolli LA (2008) Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study. J Control Release 132(1):12–20

    Article  CAS  PubMed  Google Scholar 

  • Cevc G (1997) Drug delivery across the skin. Expert Opin Investig Drugs 6(12):1887–1937

    Article  CAS  PubMed  Google Scholar 

  • Cevc G (2003) Transdermal drug delivery of insulin with ultradeformable carriers. Clin Pharmacokinet 42(5):461–474

    Article  CAS  PubMed  Google Scholar 

  • Cevc G (2004) Lipid vesicles and other colloids as drug carriers on the skin. Adv Drug Deliv Rev 56(5):675–711

    Article  CAS  PubMed  Google Scholar 

  • Cevc G, Schatzlein A, Richardsen H (2002) Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements. Biochim Biophys Acta 1564(1):21–30

    Article  CAS  PubMed  Google Scholar 

  • Clegg R (2009) Förster resonance energy transfer—FRET: what is it, why do it, and how it’s done. In: Gadella TWJ (ed) FRET and FLIM techniques, vol 33, pp 1–57

    Google Scholar 

  • Coutinho A, Garcia C, Gonzalez-Rodriguez J, Lillo MP (2007) Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET. Biophys Chem 130(1–2):76–87

    Article  CAS  PubMed  Google Scholar 

  • Daniel E, Weber G (1966) Cooperative effects in binding by bovine serum albumin. I. The binding of 1-anilino-8-naphthalenesulfonate. Fluorimetric titrations. Biochemistry 5(6):1893–1900

    Article  CAS  PubMed  Google Scholar 

  • Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C (2006) Multi-photon excitation microscopy. Biomed Eng Online 5:36

    Article  PubMed  PubMed Central  Google Scholar 

  • Digman MA, Gratton E (2012) Scanning image correlation spectroscopy. Bioessays 34(5):377–385

    Article  PubMed  PubMed Central  Google Scholar 

  • Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005a) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89(2):1317–1327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E (2005b) Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys J 88(5):L33–L36

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Digman MA, Caiolfa VR, Zamai M, Gratton E (2008) The phasor approach to fluorescence lifetime imaging analysis. Biophys J 94(2):L14–L16

    Article  CAS  PubMed  Google Scholar 

  • Eftink MR (1994) The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys J 66(2 Pt 1):482–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eftink MR, Ghiron CA (1981) Fluorescence quenching studies with proteins. Anal Biochem 114(2):199–227

    Article  CAS  PubMed  Google Scholar 

  • Gratton E, Jameson DM, Hall RD (1984) Multifrequency phase and modulation fluorometry. Annu Rev Biophys Bioeng 13:105–124

    Article  CAS  PubMed  Google Scholar 

  • Hawe A, Sutter M, Jiskoot W (2008) Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res 25(7):1487–1499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • He XM, Carter DC (1992) Atomic structure and chemistry of human serum albumin. Nature 358(6383):209–215

    Article  CAS  PubMed  Google Scholar 

  • Honeywell-Nguyen PL, Gooris GS, Bouwstra JA (2004) Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations into human skin in vivo. J Investig Dermatol 123(5):902–910

    Article  CAS  PubMed  Google Scholar 

  • Honeywell-Nguyen PL, Groenink HWW, Bouwstra JA (2006) Elastic vesicles as a tool for dermal and transdermal delivery. J Liposome Res 16(3):273–280

    Article  CAS  Google Scholar 

  • Isasi SC, Bianco ID, Fidelio GD (1995) Gangliosides raise the intracellular Ca2+ level in different cell types. Life Sci 57(5):449–456

    Article  CAS  PubMed  Google Scholar 

  • Jameson D (1998) Gregorio Weber, 1916–1997: a fluorescent lifetime. Biophys J 75:419–421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110(5):2685–2708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jameson D, Croney JC, Moens P (2003) Fluorescence: basic concepts, practical aspects and some anecdotes. Methods Enzymol 360:1–43

    Article  CAS  PubMed  Google Scholar 

  • Jameson DM, Ross JA, Albanesi JP (2009) Fluorescence fluctuation spectroscopy: ushering in a new age of enlightenment for cellular dynamics. Biophys Rev 1(3):105–118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Johnson ID (2010a) Indicators for Ca2+, Mg2+, Zn2+ and other metal ions. In: The molecular probes handbook: a guide to fluorescent probes and labeling technologies, 11th edn. Life Technologies Corporation, sect 19.12

    Google Scholar 

  • Johnson ID (2010a) The molecular probes handbook: a guide to fluorescent probes and labeling technologies, 11th edn. Life Technologies Corporation, Carlsbad

    Google Scholar 

  • Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York

    Book  Google Scholar 

  • Lakowicz JR, Weber G (1973a) Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules. Biochemistry 12(21):4161–4170

    Article  CAS  PubMed  Google Scholar 

  • Lakowicz JR, Weber G (1973b) Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale. Biochemistry 12(21):4171–4179

    Article  CAS  PubMed  Google Scholar 

  • Lasagna M, Vargas V, Jameson DM, Brunet JE (1996) Spectral properties of environmentally sensitive probes associated with horseradish peroxidase. Biochemistry 35(3):973–979

    Article  CAS  PubMed  Google Scholar 

  • Maystre D (2012) Theory of Wood’s anomalies. In: Enouch S, Bonod N (eds) Plasmonics, vol 167, Springer series in optical sciences. Springer, Berlin, pp 39–83

    Chapter  Google Scholar 

  • Murata S, Iwanaga C, Toda T, Kokubun H (1972) Fluorescence and radiationless transitions from the second excited states of azulene derivatives. Ber Bunsenges Phys Chem 76:1176–1183

    CAS  Google Scholar 

  • Newton I (1665–1666) Of colours. Cambridge University Library, Cambridge

    Google Scholar 

  • Parasassi T, Gratton E (1995) Membrane lipid domains and dynamics as detected by LAURDAN fluorescence. J Fluoresc 5(1):59–69

    Article  CAS  PubMed  Google Scholar 

  • Parasassi T, Krasnowska EK, Bagatolli LA, Gratton E (1998) LAURDAN and Prodan as polarity sensitive fluorescent membrane probes. J Fluoresc 8(4):365–373

    Article  CAS  Google Scholar 

  • Perrin F (1926) Polarisation de la lumière de fluorescence. Vie moyenne des molécules dans l'etat excité. J Phys Radium 7:390–401

    Article  CAS  Google Scholar 

  • Rosso SB, Gonzalez M, Bagatolli LA, Duffard RO, Fidelio GD (1998) Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin. Life Sci 63(26):2343–2351

    Article  CAS  PubMed  Google Scholar 

  • Simonsson C, Madsen JT, Graneli A, Andersen KE, Karlberg AT, Jonsson CA, Ericson MB (2011) A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations. Toxicol Appl Pharmacol 252(3):221–227

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Stefl M, James NG, Ross JA, Jameson DM (2011) Applications of phasors to in vitro time-resolved fluorescence measurements. Anal Biochem 410:62–69

    Article  CAS  PubMed  Google Scholar 

  • Strickler SJ, Berg RA (1962) Relationship between absorption intensity and fluorescence lifetime of molecules. J Chem Phys 37:814–822

    Article  CAS  Google Scholar 

  • Tsien CL, Fraser HS, Long WJ, Kennedy RL (1998) Using classification tree and logistic regression methods to diagnose myocardial infarction. Stud Health Technol Inform 52(Pt 1):493–497

    PubMed  Google Scholar 

  • Ustione A, Piston DW (2011) A simple introduction to multiphoton microscopy. J Microsc 243(3):221–226

    Article  CAS  PubMed  Google Scholar 

  • Valeur B, Berberan-Santos MN (2013) Molecular fluorescence. Principles and applications, 2nd edn. Wiley-VCH Verlag y Co, Weinheim

    Google Scholar 

  • van Kuijk-Meuwissen MEMJ, Junginger HE, Bouwstra JA (1998) Interactions between liposomes and human skin in vitro, a confocal laser scanning microscopy study. Biochim Biophys Acta Biomembr 1371(1):31–39

    Article  Google Scholar 

  • van Munster EB, Gadella TW (2005) Fluorescence lifetime imaging microscopy (FLIM). Adv Biochem Eng Biotechnol 95:143–175

    PubMed  Google Scholar 

  • Weber G (1952) Polarization of the fluorescence of macromolecules. I. Theory and experimental method. Biochem J 51(2):145–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weber G, Daniel E (1966) Cooperative effects in binding by bovine serum albumin. II. The binding of 1-anilino-8-naphthalenesulfonate. Polarization of the ligand fluorescence and quenching of the protein fluorescence. Biochemistry 5(6):1900–1907

    Article  CAS  PubMed  Google Scholar 

  • Weber G, Farris FJ (1979) Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry 18(14):3075–3078

    Article  CAS  PubMed  Google Scholar 

  • Weber G, Laurence DJ (1954) Fluorescent indicators of adsorption in aqueous solution and on the solid phase. Biochem J 56 (325th Meeting):xxxi

    Google Scholar 

  • Weber G, Young LB (1964) Fragmentation of bovine serum albumin by pepsin. I. The origin of the acid expansion of the albumin molecule. J Biol Chem 239:1415–1423

    CAS  PubMed  Google Scholar 

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Acknowledgments

The author wants to thanks the Danish National Research Foundation (which supports MEMPHYS-Center for Biomembrane Physics) and Drs. David Jameson and Roberto Stock for the critical reading of this manuscript.

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Correspondence to Luis A. Bagatolli .

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Bagatolli, L.A. (2016). Fluorescence Spectroscopy: Basic Foundations and Methods. In: Müllertz, A., Perrie, Y., Rades, T. (eds) Analytical Techniques in the Pharmaceutical Sciences. Advances in Delivery Science and Technology. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-4029-5_2

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