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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 intersystem crossing (dotted arrow).

Five-Step Plan Reduces Training Costs to Less than 1% of Operating Costs Issue: Response: HR at a 600-employee manufacturing firm in the South needed to be sure employees cost-effectively completed scheduled training. A training department overhaul. The company s training supervisor attributes their success to five improvements: (1) an in-house train-the-trainer program, (2) the use of instructional systems design for training, (3) networking with other firms for best practices, (4) close work with management to ensure that all training needs are met successfully, and (5) a Web-based delivery system. HR s training budget was reduced from 3.6% to 1% of plant operating costs.

The triplet state is important, because molecules with electrons in this state are chemically reactive, which can lead to photobleaching and the production of damaging free radicals (discussed later in this chapter) During fluorescence, the absorption and re-emission events occur nearly simultaneously, the interval being only 10 9 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 second 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, fluorescent 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 Fluorochromes 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 spectrum 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.

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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 lowerenergy, 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/ , where h is Planck s constant, c is the speed of light, and is the wavelength Since the photon energy is reduced during absorption and re-emission, the wavelength increases.