Fluorescence Microscopy in Life Sciences

Spectral Analysis

Author(s): Juan Carlos Stockert and Alfonso Blazquez-Castro

Pp: 135-158 (24)

DOI: 10.2174/9781681085180117010009

* (Excluding Mailing and Handling)


Studies on the spectral (electronic) properties of dyes are often needed to know qualitative and quantitative aspects of their interactions with macromolecular substrates. This Chapter deals with the description and interpretation of absorption and emission spectra from some dyes and fluorochromes used in microscopical studies. General references on fluorescence spectroscopy can be found in [1-13]. For detailed information on microscopic photometry (i.e. ray paths, equipments, adjustments, and procedures), the book by Piller [14] should be consulted. At present, confocal spectral analysis generating 2D and 3D microscopical images of biological materials offers new possibilities for research applications, mainly in the localization of antitumor drugs within living cells [15]. Some key concepts and useful tips must be taken into account for a correct interpretation of spectra. Generally, emission spectra are corrected for the Raman scatter of the solvent and for harmonics. To record adequately far red emission, the detector sensitivity must be able to capture long wavelengths with fidelity.

Keywords: Acridine orange, Bathochromic shift, Binding mechanisms, Caffeine, Clay minerals, Co-solutes, Detergent micelles, Harmonics, Hypsochromic shift, Ionic strength, Macrospectra, Molecular rigidity, Raman band, Rayleigh-Tyndall band, Viscous solvents.

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