Page 198 - Fundamentals of Light Microscopy and Electronic Imaging

PHYSICAL BASIS OF FLUORESCENCE 181

turn composed of a number of sub energy levels, which do not concern us here. There
are two categories of excited states, characterized by different spin states of the excited
electron—the singlet excited state and the triplet excited state. Most commonly, an
excited electron occupies an excitation level within the singlet excited state (straight
upward pointing arrows), and when it collapses to the ground state, energy can be given
up as fluorescence emission (straight downward pointing arrows). Alternatively, energy
can be given up as heat (internal conversion), in which case no photon is emitted (wavy
downward pointing arrows). When excited above the ground state, there is a probability
that an electron can also enter the triplet excited state through a process called intersys-
tem crossing (dotted arrow). The triplet state is important, because molecules with elec-
trons in this state are chemically reactive, which can lead to photobleaching and the
production of damaging free radicals (discussed later in this chapter). During fluores-
cence, the absorption and re-emission events occur nearly simultaneously, the interval
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being only 10 –10 12 seconds; therefore, fluorescence stops the moment there is no
more exciting incident light. The emission process is called phosphorescence if the
period between excitation and emission is not instantaneous and lasts fractions of a sec-
ond to minutes. These processes should not be confused with bioluminescence, such as
that exhibited by firefly luciferase, in which electrons are excited by chemically driven
processes rather than by absorbing external radiation.
Molecules that are capable of fluorescing are called fluorescent molecules, fluores-
cent dyes, or fluorochromes. If a fluorochrome is conjugated to a large macromolecule
(through a chemical reaction or by simple adsorption), the tagged macromolecule is said
to contain a fluorophore, the chemical moiety capable of producing fluorescence. Fluo-
rochromes exhibit distinct excitation and emission spectra that depend on their atomic
structure and electron resonance properties. Fluorescent dyes usually contain several
unconjugated double bonds. The spectra for fluorescein-conjugated immunoglobulin
(IgG) are shown in Figure 11-3. Molecules absorb light and re-emit photons over a spec-
trum of wavelengths (the excitation spectrum) and exhibit one or more characteristic
excitation maxima. Absorption and excitation spectra are distinct but usually overlap,
sometimes to the extent that they are nearly indistinguishable. However, for fluorescein
and many other dyes, the absorption and excitation spectra are clearly distinct. The
widths and locations of the spectral curves are important, particularly when selecting
two or more fluorochromes for labeling different molecules within the same specimen.
Re-emission of fluorescent light from excited dye molecules likewise occurs over a
broad spectrum of longer wavelengths (the emission spectrum) even when excitation is
performed with a monochromatic source such as a laser. This is because electrons
occupy excited states for various lengths of time during which they give up varying
amounts of vibrational energy, some of it as heat, resulting in the re-emission of lower-
energy, longer-wavelength photons over a spectrum of wavelengths. Because some
energy is given up during the process, the wavelength of a fluorescent photon is usually
longer than the wavelength of the photon exciting the molecule. It will be remembered
that the energy of a photon is given as E hc/λ, where h is Planck’s constant, c is the
speed of light, and λ is the wavelength. Since the photon energy is reduced during
absorption and re-emission, the wavelength increases. The reader is encouraged to
review the relationships between the energy, frequency, and wavelength of photons
described in Chapter 2.
Finally, the shapes of spectral curves and the peak wavelengths of absorption and
emission spectra vary, depending on factors contributing to the chemical environment of
the system, including pH, ionic strength, solvent polarity, O concentration, presence of
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