Page 200 - Fundamentals of Light Microscopy and Electronic Imaging

PROPERTIES OF FLUORESCENT DYES 183

high value—but for protein-bound fluorescein at neutral pH, the quantum efficiency is
typically 0.6–0.3.
Other important characteristics of dyes are their resistance to photobleaching, solu-
bility in aqueous media, and chemical stability. Quenching and photobleaching reduce
the amount of fluorescence and are of great practical significance to the microscopist.
Quenching reduces the quantum yield of a fluorochrome without changing its fluores-
cence emission spectrum and is caused by interactions with other molecules including
other fluorochromes. Conjugation of fluorescein to a protein usually causes a significant
reduction in the quantum yield because of charge-transfer interactions with nearby aro-
matic amino acids. Proteins such as IgG or albumin that are conjugated with 5 or more
fluorescein molecules, for example, fluoresce less than when bound to 2–3 molecules,
because energy is transferred to nonfluorescent fluorescein dimers. Photobleaching
refers to the permanent loss of fluorescence by a dye due to photon-induced chemical
damage and covalent modification. As previously discussed, photobleaching occurs
when a dye molecule, excited to one of its electronic singlet states, transits to a triplet
excited state (Fig. 11-2). Molecules in this state are able to undergo complex reactions
with other molecules. Reactions with molecular oxygen permanently destroy the fluo-
rochrome and produce singlet oxygen species (free radicals) that can chemically mod-
ify other molecules in the cell. Once the fluorochrome is destroyed, it usually does not
recover. The rate of photobleaching can be reduced by reducing the excitation or lower-
ing the oxygen concentration. Methods for reducing oxygen concentration as a way to
protect live cells are described at the end of the chapter.

Demonstration: Fluorescence of Chlorophyll and Fluorescein

The phenomena and principles of fluorescence can be demonstrated using a fil-
tered alcoholic extract of spinach leaves and an aqueous solution of fluorescein.
The procedures for preparing these samples are given in Appendix II.
Although an extract of spinach contains a mixture of chlorophylls a and b,
plus carotene and xanthophyll, most of the fluorescence phenomena are due to
chlorophyll a. The fluorescence spectra of chlorophyll a, the major chlorophyll
component, are shown in Figure 11-4. The figure shows that the absorption/exci-
tation spectrum is broad and distinctly bimodal, with absorption peaks widely
separated in the blue and red portions of the visible spectrum. Fluorescence emis-
sion at red wavelengths with an emission peak at 670 nm is the same regardless
of whether blue or red wavelengths are used to excite the molecule. Notice too the
very large Stokes shift from 420 to 670 nm—a difference of 250 nm. Normally
the chlorophyll and other pigments in intact chloroplasts act as energy transduc-
ers and do not fluoresce; rather, the electrons excited by absorbed photons are
transferred to nearby enzyme assemblies (photosystems I and II) for the fixation
of carbon dioxide into carbohydrate. However, when chlorophyll is extracted into
a soluble form in alcohol, no electron transfer is possible, and absorbed photons
are re-emitted as deep red fluorescence.
If the flask of chlorophyll is placed over a bright white light source, a deep
emerald green color is seen. Green wavelengths are observed because red and blue
wavelengths are selectively absorbed. (Refer to subtraction colors in Chapter 2.)