Molecular Fluorescence

1.5 Molecular fluorescence as an analytical tool 15

moreover it is sensitive to pH. If the OH group is replaced by Oa(CH2)n aCH3, the resulting molecule becomes pH-insensitive and amphiphilic; the fluorophore moiety plays the role of a polar head and thus is located at the surfactant–water interface of systems consisting of amphiphilic molecules (bilayers of membranes and vesicles, micellar systems, etc.). Conversely, the pyrene moiety of pyrenedodecanoic acid is deeply embedded in the hydrophobic part of an organized assembly. 1,6-Diphenyl-1,3,5-hexatriene (DPH) is located in the hydrocarbon region of bilayers of membrane and vesicles, whereas its cationic analog TMA–DPH is anchored with its charged group at the surfactant–water interface. The latter is thus a probe of the upper region of bilayers. Cisand trans-parinaric acids are good examples of probes causing minimum spatial perturbation to organized assemblies.

The above examples show that a very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). On the other hand, insensitivity to the chemical nature of the environment is preferable in some cases (e.g. in fluorescence polarization or energy transfer experiments). Environment-insensitive probes are also better suited to fluorescence microscopy and flow cytometry.

A criticism often aimed at the use of extrinsic fluorescent probes is the possible local perturbation induced by the probe itself on the microenvironment to be probed. There are indeed several cases of systems perturbed by fluorescent probes. However, it should be emphasized that many examples of results consistent with those obtained by other techniques can be found in the literature (transition temperature in lipid bilayer, flexibility of polymer chains, etc.). To minimize the perturbation, attention must be paid to the size and shape of the probe with respect to the probed region.

In conclusion, the choice of a fluorescent probe is crucial for obtaining unambiguous interpretations. The major aspects that should be taken into consideration are shown in Figure 1.6.

1.5

Molecular fluorescence as an analytical tool

Analytical techniques based on fluorescence detection are very popular because of their high sensitivity and selectivity, together with the advantages of spatial and temporal resolution, and the possibility of remote sensing using optical fibers.

When an analyte is fluorescent, direct fluorometric detection is possible by means of a spectrofluorometer operating at appropriate excitation and observation wavelengths. This is the case for aromatic hydrocarbons (e.g. in crude oils), proteins (e.g. in blood serum, in cow milk), some drugs (e.g. morphine), chlorophylls, etc. Numerous fields of applications have been reported: analysis of air and water pollutants, oils, foods, drugs; monitoring of industrial processes; monitoring of species of clinical relevance; criminology; etc.

However, most ions and molecules are not fluorescent and the main indirect methods that are used in this case are the following:

16 1 Introduction

Fig. 1.6. Strategy for the choice of a fluorescent probe. Dn, F, and t are the Stokes shift, quantum yield and lifetime, respectively (see definitions in Chapter 3).

.Derivatization, i.e. reaction of the analyte with a reagent leading to a fluorescent compound, is often used in conjunction with liquid chromatography with fluorescence detection. This method is currently used in biochemistry and clinical

. chemistry.Formation of a fluorescent complex is the basis of most methods of ion and mole- cule recognition (see Chapter 9).

. Fluorescence quenching resulting from the collision of the analyte with a fluorescent compound (see Chapter 4). This method is particularly well suited to the detection of gases such as oxygen (dissolved in water or blood), SO2, H2S, ammonia, HCl, Cl2, chlorocarbons, etc.

Finally, fluorescence immunoassay is a method of major importance for biochemical and biomedical applications.

1.6

Ultimate temporal and spatial resolution: femtoseconds, femtoliters, femtomoles and single-molecule detection

The ability of fluorescence to provide temporal information is of major importance. Great progress has been made since the first determination of an excited-state lifetime by Gaviola in 1926 using a phase fluorometer. A time resolution of a few tens of picosecond can easily be achieved in both pulse and phase fluorometries by using high repetition rate picosecond lasers and microchannel plate photo-

1.6 Ultimate temporal and spatial resolution 17

multipliers (see Chapter 6). Such a time resolution is limited by the response of the photomultiplier but not by the width of the laser pulse, which can be as short as 50–100 fs (1 femtosecond ¼ 10 15 second) (e.g. with a titanium:sapphire laser). The time resolution can be reduced to a few picoseconds with a streak camera. To get an even better time resolution (100–200 fs), a more recent technique based on fluorescence up-conversion has been developed (see Chapter 11).

Regarding spatial resolution, fluorescence microscopy in confocal configuration or with two-photon excitation (see Chapter 11) allows the di raction limit to be approached, which is approximately half the wavelength of the excitation light (0.2–0.3 mm for visible radiation) with the advantage of three-dimensional resolution. The excitation volume can be as small as 0.1 fL (femtoliter). Compared to conventional fluorometers, this represents a reduction by a factor of 1010 of the excitation volume. At high dilution (A10 9 M or less), fluorophores entering and leaving such a small volume cause changes in fluorescence intensity. Analysis of these fluctuations (which is the object of fluorescence correlation spectroscopy; see Chapter 11) in terms of autocorrelation function can provide information on translational di usion, flow rates and molecular aggregation. Fluctuations can also be caused by chemical reactions or rotational di usion. The typical lower limit concentration is @1 fM (femtomol L 1). The progress of these techniques allows us to study molecular interactions at the unsurpassed sensitivity of single-molecule detection.

The di raction limit can be overcome by using a sub-wavelength light source and by placing the sample very close to this source (i.e. in the near field). The relevant domain is near-field optics (as opposed to far-field conventional optics), which has been applied in particular to fluorescence microscopy. This technique, called near-field scanning optical microscopy (NSOM), is an outstanding tool in physical, chemical and life sciences for probing the structure of matter or living systems. The resolution is higher than in confocal microscopy, with the additional capability of force mapping of the surface topography, and the advantage of reduced photobleaching. Single molecule detection is of course possible by this technique.

The first optical detection of a single molecule was reported in 1989 by Moerner and Kador, who detected a single pentacene molecule doped into a p-terphenyl crystal (at liquid helium temperature) using absorption with a double modulation technique. Fluorescence excitation spectroscopy on a single molecule was demonstrated for the first time by Orrit and Bernard in 1990. The detection of a single fluorescent molecule in solution was achieved not much later. Therefore, Schro¨- dinger’s statement (in 1952) has been outspaced by reality: ‘. . . we never experiment with just one electron or atom or molecule. In thought experiments we sometimes assume we do, this invariably entails ridiculous consequences.’

Single molecule detection o ers the possibility of selecting, trapping, sorting, picking, and even manipulating molecules, especially biological macromolecules. Detection and spectroscopy of individual fluorescent molecules thus provide new tools not only in basic research but also in biotechnology and pharmaceutical industries (e.g. drug screening).

22 2 Absorption of UV–visible light

Fig. 2.2. Distinction between singlet and triplet states, using formaldehyde as an example.

plicities of both the ground and excited states ðM ¼ 2S þ 1Þ is equal to 1, both are called singlet state (usually denoted S0 for the ground state, and S1; S2; . . . for the excited states) (Figure 2.2)1). The corresponding transition is called a singlet–singlet transition. It will be shown later that a molecule in a singlet excited state may undergo conversion into a state where the promoted electron has changed its spin; because there are then two electrons with parallel spins, the total spin quantum number is 1 and the multiplicity is 3. Such a state is called a triplet state because it corresponds to three states of equal energy. According to Hund’s Rule, the triplet state has a lower energy than that of the singlet state of the same configuration.

In a molecule such as formaldehyde, the bonding and non-bonding orbitals are localized (like the bonds) between pairs of atoms. Such a picture of localized orbitals is valid for the s orbitals of single bonds and for the p orbitals of isolated double bonds, but it is no longer adequate in the case of alternate single and double carbon–carbon bonds (in so-called conjugated systems). In fact, overlap of the p orbitals allows the electrons to be delocalized over the whole system (resonance

1)In some cases, the ground state is not a singlet state, e.g. dioxygen, anion and cation radicals of aromatic molecules.