Fluorescence quenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching. These include excited state reactions, energy transfer, complex formation, and collisional quenching. In this chapter we will be concerned primarily with quenching resulting from collisional encounters between the fluorophore and quencher, which is called collisional or dynamic quenching. We will also consider static quenching, which is due to complex formation. Static quenching is a frequent complicating factor in the analysis of dynamic quenching. In addition to the processes described above, apparent quenching can occur due to the optical properties of the sample. For example, high optical densities or turbidity can result in decreased fluorescence intensities. This is a trivial type of quenching which contains little molecular information. Throughout this chapter we will assume such trivial effects are not the cause of the observed decreases in fluorescence intensity.

Quenching of Fluorescence

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD-MA), a second-generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet-state, singlet oxygen (O2 (1 delta g)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin-type molecule shows a strong absorbance in the red (lambda approximately 688 nm, epsilon approximately 33,000 M-1 cm-1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, Es = 42.1 kcal mol-1, lifetime, tau f = 5.2 ns and fluorescence quantum yield, phi f = 0.05 in air-saturated solution. The triplet state of BPD-MA has a lifetime, tau T > or = 25 microseconds, an energy level, ET = 26.9 kcal mol-1 and the molar absorption coefficient is epsilon T = 26,650 M-1 cm-1 at 720 nm. A dramatic effect of oxygen on the fluorescence (phi f) and intersystem crossing (phi T) quantum yields has been observed. The BPD-MA presents rather high triplet (phi T = 0.68 under N2-saturated conditions) and singlet oxygen (phi delta = 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (phi Pb) of which was found to be on the order of 5 x 10(-5) in organic solvents.

Photophysical and photosensitizing properties of benzoporphyrin derivative monoacid ring A (BPD-MA)

Solute fluorescence quenching reactions were first applied to biochemical problems in the late 1960s and early 1970s, 7) and since that time they have been a very valuable research tool for studies with proteins, membranes, and other macromolecular assemblies. Quenching reactions are easy to perform, require only a small sample, usually are nondestructive, and can be applied to almost any system that has an intrinsic or extrinsic fluorescence probe. The most important characteristic, however, is the value of the information that these reactions can provide. Solute quenching reactions, using quenchers such as molecular oxygen, acrylamide, or iodide ion, provide information about the location of fluorescent groups in a macromolecular structure. A fluorophore that is located on the surface of a larger structure will be relatively accessible to a solute quencher that is dissolved in the aqueous phase. A fluorophore that is removed from the surface of a structure will be quenched to a lesser degree by the quencher. Thus, the quenching reaction can be used to probe topographical features of a macromolecular assembly and to sense any structural changes that may be caused by varying conditions or the addition of reagents. In addition, quenching reactions can, in some situations, provide information about conformational fluctuations. In Sections 2.3 and 2.4 I will discuss several examples of the use of solute quenchers in studies with proteins, membranes, and nucleic acids. Solute fluorescence quenching reactions can also be used to selectively alter the fluorescence properties of a sample in order to resolve contributions or aid in the measurement of data. To elaborate on this point, consider the different characteristics of fluorescence: the quantum yield, excitation and

Fluorescence Quenching: Theory and Applications

— The values of the absolute quantum yields of fluorescence and of intersystem crossing have been obtained for porphin and its derivatives by methods based on relative measurements involving flash photolysis. For a given compound the sum of these quantum yields is unity within experimental error which shows that the energetics of the molecules investigated is determined by competition of only two processes, viz., fluorescence and intersystem crossing, while internal conversion plays a negligible role. The values of the corresponding transition probabilities have been determined with the help of phase-fluorometric measurements. The dependence of the transition probabilities on molecular structure is discussed.

Energetics of photophysical processes in chlorophyll-like molecules.

Optical sensing of analytes is often accomplished using fluorescence detection. Quantitation is usually performed by measuring the fluorescence of a probe whose intensity changes in response to the analyte of interest. In the present report we describe sensing based on changes in lifetime or decay time of the probe. Such a sensing scheme is advantageous because, in contrast to intensity measurements, the decay times are mostly independent of intensity changes due to light losses, photobleaching or probe washout. Present technology allows nanosecond (ns) decay times to be measured by the phase-modulation method with inexpensive and robust instrumentation. In particular, we describe lifetime-based pH sensing using a green 543 nm He-Ne laser, sensing of Ca2+ using 514 nm excitation and a Ca2+ competitive assay for glucose using an energy-transfer mechanism. We also discuss emerging applications of phase-modulation method sensing to cellular imaging and flow cytometry.

Fluorescence lifetime-based sensing of pH, Ca2+, K+ and glucose

The solubility of xenon in 95 % ethanol is shown to be sufficient to quench the fluorescence of aromatic hydrocarbons with high efficiency. Quenching is due to catalyzed intersystem crossing from the first excited singlet state eventually producing the lowest triplet state. Quenching constants are given for nine molecules and the quantum yields ϕT of triplet state production of pyrene and 9-phenylanthracene in 95 % ethanol have been measured. Xenon gas is shown to be an ideal fluorescence quencher for determining ϕT in solution.

K. Tickle

Papers

Mechanism of fluorescence quenching in solution. Part 2.—Quenching by xenon and intersystem crossing efficiencies