Excited-state lifetime molecules

Pulsed method. Using a pulsed or modulated excitation light source instead of constant illumination allows investigation of the time dependence of emission polarization. In the case of pulsed excitation, the measured quantity is the time decay of fluorescent emission polarized parallel and perpendicular to the excitation plane of polarization. Emitted light polarized parallel to the excitation plane decays faster than the excited state lifetime because the molecule is rotating its emission dipole away from the polarization plane of measurement. Emitted light polarized perpendicular to the excitation plane decays more slowly because the emission dipole moment is rotating towards the plane of measurement. 

Exciplexes are complexes of the excited fluorophore molecule (which can be electron donor or acceptor) with the solvent molecule. Like many bimolecular processes, the formation of excimers and exciplexes are diffusion controlled processes. The fluorescence of these complexes is detected at relatively high concentrations of excited species, so a sufficient number of contacts should occur during the excited state lifetime and, hence, the characteristics of the dual emission depend strongly on the temperature and viscosity of solvents. A well-known example of exciplex is an excited state complex of anthracene and /V,/V-diethylaniline resulting from the transfer of an electron from an amine molecule to an excited anthracene. Molecules of anthracene in toluene fluoresce at 400 nm with contour having vibronic structure. An addition to the same solution of diethylaniline reveals quenching of anthracene accompanied by appearance of a broad, structureless fluorescence band of the exciplex near 500 nm (Fig. 2 )... 

Molecular emission is referred to as luminescence or fluorescence and sometimes phosphorescence. While atomic emission is generally instantaneous on a time scale that is sub-picoseconds, molecular emission can involve excited states with finite, lifetimes on the order of nanoseconds to seconds. Similar molecules can have quite different excited state lifetimes and thus it should be possible to use both emission wavelength and emission apparent lifetime to characterize molecules. The instrumental requirements will be different from measurements of emission, only in detail but not in principles, shared by all emission techniques. 

Lochmuller and coworkers used the formation of excimer species to answer a distance between site question related to the organization and distribution of molecules bound to the surface of silica xerogels such as those used for chromatography bound phases. Pyrene is a flat, poly aromatic molecule whose excited state is more pi-acidic than the ground state. An excited state of pyrene that can approach a ground state pyrene within 7A will form an excimer Pyr +Pyr (Pyr)2. Monomer pyrene emits at a wavelength shorter than the excimer and so isolated versus near-neighbor estimates can be made. In order to do this quantitatively, these researchers turned to measure lifetime because the monomer and excimer are known to have different lifetimes in solution. This is also a way to introduce the concept of excited state lifetime. 

Fluorescence quenching is described in terms of two mechanisms that show different dependencies on quencher concentration. In dynamic quenching, the quencher can diffuse at least a few nanometers on the time scale of the excited state lifetime (nanoseconds). In static quenching, mass diffusion is suppressed. Only those dye molecules which are accidentally close to a quencher will be affected. Those far from a quencher will fluoresce normally, unaware of the presence of quenchers in the system. These processes are described below for the specific case of PMMA-Phe quenched by MEK. 

Montalti and co-workers studied dansyl [27] and pyrene [28] derivatives and found the fluorescence quantum yields and excited-state lifetime of these two dyes increased in DDSNs. They attributed the enhancements to the shielding effect from the quenchers or polar solvent in the suspension. Their studies also demonstrated that the lifetime of the doped dye molecules was also dependent on the size of the DDSNs. Small DDSNs had a larger population of the short-living moieties that were more sensitive to the environment outside the DDSN. In contrast, the large DDSN had a larger population of the long-living moieties that were not sensitive to the environment. 

Here, y, is a factor of (1 + Fm/F). In a fluorescence enhancement system, interactions between the metal and dye molecules result in shortening of the excited-state lifetime, thus improving the photostability of the dye. 

Following an external perturbation, the fluorescence quantum yield can remain proportional to the lifetime of the excited state (e.g. in the case of dynamic quenching (see Chapter 4), variation in temperature, etc.). However, such a proportionality may not be valid if de-excitation pathways - different from those described above - result from interactions with other molecules. A typical case where the fluorescence quantum yield is affected without any change in excited-state lifetime is the formation of a ground-state complex that is non-fluorescent (static quenching see Chapter 4). 

In Chapter 3 we described the intrinsic pathways of de-excitation of a molecule M the sum fey of the rate constants for these processes is equal to the reciprocal of the excited-state lifetime to1 ... 

When M and Q cannot change their positions in space relative to one another during the excited-state lifetime of M (i.e. in viscous media or rigid matrices), Perrin proposed a model in which quenching of a fluorophore is assumed to be complete if a quencher molecule Q is located inside a sphere (called the sphere of effective quenching, active sphere or quenching sphere) of volume Vq surrounding the fluorophore M. If a quencher is outside the active sphere, it has no effect at all on M. Therefore, the fluorescence intensity of the solution is decreased by addition of Q, but the fluorescence decay after pulse excitation is unaffected. 

The excited-state lifetime of the uncomplexed fluorophore M is unaffected, in contrast to dynamic quenching. The fluorescence intensity of the solution decreases upon addition of Q, but the fluorescence decay after pulse excitation is unaffected. Quinones, hydroquinones, purines and pyrimidines are well-known examples of molecules responsible for static quenching. 

When the two monomers are linked by a short flexible chain, intramolecular excimers can be formed. This process is still diffusion-controlled, but in contrast to the preceding case, it is not translational it requires a close approach between the two molecules via internal rotations during the excited-state lifetime. Equations (4.44), (4.45), (4.47) to (4.49) are still valid after replacing k [M] by k because intramolecular excimer formation is independent of the total concentration. Estimation of the local fluidity of a medium can be achieved by means of probes capable of forming intramolecular excimers (see Chapter 8). 

If excited molecules can rotate during the excited-state lifetime, the emitted fluorescence is partially (or totally) depolarized (Figure 5.9). The preferred orientation of emitting molecules resulting from photoselection at time zero is indeed gradually affected as a function of time by the rotational Brownian motions. From the extent of fluorescence depolarization, we can obtain information on the molecular motions, which depend on the size and the shape of molecules, and on the fluidity of their microenvironment. 

The anthroyl fluorophore is located deep in the hydrophobic region of the lipid bilayer corresponding to the C9-C16 segment of the acyl chains. The excited-state lifetime, associated with a non-structured fluorescence spectrum with a maximum at 460 nm (to be compared to those shown in Figure 7.6), can be accounted for by interaction of the fluorophore with water molecules that diffuse across the bilayer. Information is thus obtained on the permeability of lipid bilayers to water and its modulation by cholesterol. 

The point is now to estimate the maximum number of photons that can be detected from a burst. The maximum rate at which a molecule can emit is roughly the reciprocal of the excited-state lifetime. Therefore, the maximum number of photons emitted in a burst is approximately equal to the transit time divided by the excited-state lifetime. For a transit time of 1 ms and a lifetime of 1 ns, the maximum number is 106. However, photobleaching limits this number to about 105 photons for the most stable fluorescent molecules. The detection efficiency of specially designed optical systems with high numerical aperture being about 1%, we cannot expect to detect more than 1000 photons per burst. The background can be minimized by careful dean-up of the solvent and by using small excitation volumes ( 1 pL in hydrodynamically focused sample streams, 1 fL in confocal exdtation and detection with one- and two-photon excitation, and even smaller volumes with near-field excitation). 

One can expect that the analysis of continuous distributions of electronic excited-state lifetimes will not only provide a higher level of description of fluorescence decay kinetics in proteins but also will allow the physical mechanisms determining the interactions of fluorophores with their environment in protein molecules to be elucidated. Two physical causes for such distributions of lifetimes may be considered ... 

Both the physics and the chemistry of proximity to a surface can alter the excited-state lifetime and rotational motion of a fluorescent molecule. An extrinsic label attached to BSA has been found to reduce its fluorescence lifetime upon BSA adsorption to fused silica.(95) The decrease is too large to arise from the physical near-field proximity effects discussed in Section 7.3 ... 

Because of such difficulties as the featureless absorption and emission spectra in the vacuum ultraviolet region, very weak and energy-dependent fluorescence intensity, short excited-state lifetime, etc. the photophysics and photochemistry of alkanes is much less known than those of other organic molecules, for instance, aromatic hydrocarbons. In this chapter, the present status was reviewed. 

Secondly, the emission of a particular complex species is characteristic of that species and can be used to identify the species present. Particularly is this so if excited state lifetimes are measured, as these vary dramatically depending on the number of OH groups coordinated to the Eu3+ or Tb3+ ion. This is because multiple excitation of the OH stretching mode provides an alternative deexcitation route. Measurement of lifetimes thus can be used to determine the number of coordinated water molecules.218... 

Excitation spectra have been of considerable use recently in studying both hydration numbers (by lifetime measurements) and inner-sphere complexation by anions (by observing appearance of the characteristic frequencies for e.g. the Eu3+ 5D0-+ 7F0 transition for the different possible species). Thus using a pulsed dye laser source, it was possible to demonstrate the occurrence of inner sphere complexes of Eu3+ with SCN, CI or NO3 in aqueous solution, the K values being 5.96 2, 0.13 0.01 and 1.41 0.2 respectively. The CIO4 ion did not coordinate. Excited state lifetimes suggest the nitrate species is [Eu(N03)(HzO)6,s o.4]2+ the technique here is to compare the lifetimes of the HzO and the corresponding D20 species, where the vibrational deactivation pathway is virtually inoperative.219 The reduction in lifetime is proportional to the number of water molecules complexed.217 218... 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

---