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Fluorescence Spectroscopy: Basic Foundations and Methods

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Part of the Advances in Delivery Science and Technology book series (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.

Keywords

  • Light absorption, fluorescence emission
  • Fluorescence lifetime
  • Fluorescence polarization
  • FRET
  • Fluorescence instrumentation
  • Fluorophores
  • Raster imaging correlation spectroscopy

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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.

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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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