Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Light in Biology
Fluorescence and Phosphorescence

A molecule in an electronically excited state can return to its ground state and dissipate its energy in several ways. One of these is the emission of a light quantum (fluorescence) [51–55]. The intensity and SPECTRAL PROPERTIES OF fluorescence emission are determined by illuminating a sample placed in a four-sided transparent cuvette and positioning a photomultiplier at a right angle to the exciting light beam. While absorption spectrophotometry measures The ratio of the light beam intensities before and after passing through the sample, fluorescence spectroscopy measures the absolute intensity of the emitted light. Although this intensity is low, the sensitivity of the method is considerably higher than that of Light absorption measurements. Consequently, fluorescence spectroscopy is a vital analytical technique (see, for example, Chapter 2, Section 3.5). It also proves to be an invaluable and highly sensitive tool for investigating The chemical properties of electronically excited states. Figures 13-15 present the fluorescence excitation spectra¹⁾ (which closely match the absorption spectra) for two compounds—flavin [56] and Tryptophan—along with their corresponding emission spectra. The latter are normalized so that the peak height coincides with that of the absorption spectra. Note that the fluorescence spectrum is shifted toward lower energies relative to the absorption spectrum, and that the two spectra overlap only slightly. Furthermore, the shape of the emission spectrum closely mirrors the reflection of the absorption spectrum. To understand the underlying reason for this, let us refer to the diagram in Figure 13-16. Recall that upon absorbing a light quantum, a molecule typically transitions to higher vibrational sublevels of the excited electronic state than those it occupied in the ground state. However, before a measurable amount of light can be emitted, the excess vibrational energy is dissipated, leaving the excited molecule at the lowest vibrational sublevel of the excited electronic state. It is primarily from this level that the molecule transitions back to the ground state, emitting the bulk of the light. Additionally, one must account for the fact that prior to absorption, the molecule typically resides in the lowest vibrational state of the electronic ground state, whereas fluorescence populates many excited vibrational sublevels of the ground electronic state (Fig. 13-16). Thus, as the figure shows, the fluorescence spectrum consists of a series of vibrational sub-bands with lower energies than the bands in the absorption spectrum. The only feature shared by both spectra is the 0–0 band. In reality, as seen in Figure 13-15, even the 0–0 bands do not completely coincide in most cases: in the fluorescence spectrum, this band is slightly shifted toward lower energies. This shift occurs because during or immediately after photon absorption, the solvent molecules rearrange around the absorbing molecule, settling into a more energetically favorable position. Much like the dissipation of excess vibrational energy in the excited state, the relaxation of solvent molecules around the excited chromophore leads to a more stable energy state. A similar relaxation process takes place in the ground state immediately following photon emission, contributing to the shift of the 0–0 band in the fluorescence spectrum ([52], p. 13).

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FIG. 13-15. A. Emission and excitation spectra of riboflavin tetrabutyrate in n-heptane (see text) [56]. Riboflavin tetrabutyrate concentration ~0.4 mg·l^-1. I — excitation spectrum; emission observed at 525 nm. II — emission spectrum; excitation wavelength 345 nm. B. Spectra of indole in cyclohexane at T = 196 °C [54]. I — excitation spectrum; II — fluorescence spectrum; III — phosphorescence spectrum.

FIG. 13-16. Potential energy diagrams of an organic molecule in solution in the ground (S₀), first excited singlet (S₁), and triplet (T₁) states ([5], p. 274). G — intersection point of the S₁ and T₁ curves. For clarity, the intermolecular distances r are chosen such that r_s0 < r_s1 < r_T1, which leads to spectral stretching. In reality, for complex and nearly symmetrical molecules, r_S0 ≃ r_s1 < r_T1, meaning that the 0–0 absorption and fluorescence bands almost coincide, and only the phosphorescence band is strongly shifted toward longer wavelengths.

¹⁾ The excitation spectrum is an example of an action spectrum, which represents the dependence of a system parameter on the wavelength of absorbed light. At very low concentrations of pure compounds, action spectra practically coincide with absorption spectra. However, because the value of the parameter (in this case, fluorescence intensity) is proportional to The amount of light absorbed, action spectra must be compared with plots of 1–T versus wavelength (T being transmittance; see Section B,1) rather than plots of ε versus λ. At low concentrations, these quantities are proportional. This topic is thoroughly covered in Clayton's book [51].

Figure 13-16 illustrates another characteristic of excited molecules that affects their reactivity. They can transition from the excited singlet state to a lower-energy triplet state featuring two unpaired electrons, endowing the molecule with biradical-like properties. The triplet state is long-lived and largely dictates the photochemical properties of molecules. The transition from this state back to the ground state is accompanied by phosphorescence—a delayed emission of light, as illustrated in the same figure.

Why do some molecules fluoresce while others do not? Fluorescent capability is largely determined by the radiative lifetime τ_r, which, according to equation (13-13), is related to the first-order rate constant governing the exponential decay of excited molecules via fluorescence:

The radiative lifetime depends on the wavelength and the oscillator strength of the corresponding transition. For molecules absorbing in the near-UV region, the following approximation is often used:

Thus, at ε = 10,000, the radiative lifetime (the time required for the fluorescence intensity to decrease to 1/ε of its initial value) is ~10^-8 s (10 ns). The stronger the absorption, the shorter the radiative lifetime, and vice versa. Besides fluorescence, there are alternative pathways for de-excitation; consequently, the shorter the radiative lifetime, the higher the probability of observing fluorescence.

Processes competing with fluorescence include internal conversion, intersystem crossing (which transitions the molecule to a triplet state), and photochemical reactions that the molecule may undergo while in the excited singlet state. Internal conversion is a process whereby a molecule transitions from the lowest vibrational sublevel of a higher electronically excited state to one of the high vibrational sublevels of the ground state. This process serves as the primary pathway for electronic de-excitation and directly competes with fluorescence. Therefore, the lifetime of a molecule in the excited state¹⁾ (τ) is typically shorter than τ_r. The fluorescence quantum yield is defined as

For strongly fluorescing molecules such as riboflavin, φ_f sometimes exceeds 0.25 [58].

The number of non-radiative transitions can be increased by adding quenchers. Fluorescence quenching can occur via various mechanisms, most commonly through the collision of an excited chromophore with a quencher molecule. Certain substances are exceptionally effective quenchers (e.g., iodide ions). The fluorescence quantum yield in the absence of a quencher can be expressed in terms of the rate constants for fluorescence (k_f), non-radiative transition (k_d), and phosphorescence (k_p):

In the presence of a quencher Q, The rate of transition from the excited state to the ground state increases. The ratio of the fluorescence quantum yields in the absence (φ⁰_f) and presence of the quencher is given by the Stern–Volmer equation:

The constant K is known as the Stern–Volmer quenching constant.

An example of applying fluorescence quenching to protein research is The Use of acrylamide as a quencher to determine the solvent accessibility of tryptophan side chains [59, 60].

¹⁾ The directly measured excited-state lifetime for riboflavin-5'-phosphate (ε_max = 12,200 at 450 nm) at 25 °C is ~5 ns [57].