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Photostability of Fluorescent Dyes for Single-Molecule Spectroscopy: Mechanisms and Experimental Methods for Estimating Photobleaching in Aqueous Solution

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Applied Fluorescence in Chemistry, Biology and Medicine

Abstract

Laser-induced fluorescence detection is used for various ultrasensitive analytical techniques in chemistry, biology, and medicine by probing reagents that are either autofluorescing or tagged with a fluorescent dye molecule [1–29]. Applying different microscopic techniques with tight spatial and spectral filtering, various groups have directly visualized a variety of single fluorescent dye molecules (rhodamines [5–7,15,16,30–32] and coumarins [17,18,33]) dissolved in liquids by using coherent one- and two-photon excitation. Due to the high sensitivity and specificity, fluorescence is probably the most important optical readout mode in biological, scanning confocal microscopy. These unique features of fluorescence are critically dependent on the availability of appropriate fluorophores [34–40]. Photophysical parameters in general, and photobleaching or the photostability in particular, play a very important role not only for the accuracy of single-molecule detection (SMD) by laser-induced fluorescence [17,18,41–44] and in dye laser chemistry [45,46], but in practically all applications of fluorescence spectroscopy, where a high sensitivity or a high signal rate is crucial [47]. Key properties for the suitability of a fluorescent dye are its absorption coefficient, fluorescence-, triplet-, and photobleaching quantum yield [17,18,41–44,47–53]. Photobleaching is a dynamic, mostly irreversible process in which fluorescent molecules undergo photoinduced chemical destruction upon absorption of light, thus losing their ability to fluoresce.

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Authors
  1. C. Eggeling
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  2. J. Widengren
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  3. R. Rigler
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  4. C. A. M. Seidel
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Eggeling, C., Widengren, J., Rigler, R., Seidel, C.A.M. (1999). Photostability of Fluorescent Dyes for Single-Molecule Spectroscopy: Mechanisms and Experimental Methods for Estimating Photobleaching in Aqueous Solution. In: Applied Fluorescence in Chemistry, Biology and Medicine. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-59903-3_10

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