Spectroscopy fluorescence excitation

In the visible and ultraviolet regions a very high sensitivity can be achieved, if the absorption of laser photons is monitored through the laser-induced fluorescence (Fig. 1.23). When the laser wavelength Al is tuned to an absorbing molecular transition Ei Ek, the number of photons absorbed per second along the path length Ax is 

The number of fluorescence photons emitted per second from the excited level Ek is 

8) to all levels with Em Ek. The quantum efficiency of the excited state T]k = Ak/(Ak Rk) gives the ratio of the spontaneous transition rate to the total deactivation rate, which may also include the radiationless transition rate Rk (e.g., collision-induced transitions). For = 1, the number wpi of fluorescence photons emitted per second equals the number of photons absorbed per second under stationary conditions. 

Unfortunately, only the fraction 8 of the fluorescence photons emitted into all directions can be collected, where 8 = d 2/An depends on the solid angle d 2, accepted by the fluorescence detector. Not every photon impinging onto the photomultiplier cathode releases a photoelectron only the fraction = Wpe/ ph of these photons produces on the average h q photoelectrons. The quantity r ph is called the quantum efficiency of the photocathode (Vol. 1, Sect. 4.5.2). The number npe of photoelectrons counted per second is then 

Example 1.11 Modern photomultipliers reach quantum efficiencies of 7ph = 0.2. With carefully designed optics it is possible to achieve a collection 

Doppler-Limited Absorption and Fluorescence Spectroscopy with Lasers 

Inserting this figure for npE into (6.31) illustrates that with ab- 

The attainable signal-to-noise ratio (SNR) for single molecule detection in a solid using fluorescence excitation can be approximated by the following expression [71]  

Assiuning the collection efficiency D is maximized, Eq. 4 shows that there are several physical parameters which must be chosen carefully in order to maximize the SNR. First, as stated above, the values of and fp should be as large as possible, and the laser spot should be as small as possible. The power Pp cannot be increased arbitrarily because saturation causes the peak absorption cross section to drop from its low-power value Op according to [72] 

Upon close examination of an individual single-molecule peak at lower intensity (Fig. 8(c)), the lifetime-limited homogeneous linewidth of 7.8 0.2 MHz can be observed [73]. This linewidth is also termed quantum-limited , since the optical linewidth has reached the minimum value allowed by the lifetime of the optical excited state. This value is in excellent agreement with previous photon echo mea- 

It is instructive at this point to compare the signal-to-noise ratio for SFS (TV 1) to that for one single molecule (Eq. 4). Defining TVh as the number of molecules with resonance frequency within one homogeneous width of the laser frequency, and recalling that the SFS signal excursions scale as the square root of the number of molecules in resonance (see Fig. 3), 

Electronic transitions in molecules in supersonic jets may be investigated by intersecting the jet with a tunable dye laser in the region of molecular flow and observing the total fluorescence intensity. As the laser is tuned across the absorption band system a fluorescence excitation spectrum results which strongly resembles the absorption spectrum. The spectrum 

It might be thought that the small number of molecules in a typical supersonic jet or beam would seriously limit the sensitivity of observation of the spectra. Flowever, the severe rotational cooling which may be produced results in a collapsing of the overall intensity of a band into many fewer rotational transitions. Vibrational cooling, which greatly increases the population of the zero-point level, concentrates the intensity in few vibrational transitions, and these two effects tend to compensate for the small number of molecules. 

In a skimmed supersonic jet, the parallel nature of the resulting beam opens up the possibility of observing spectra with sub-Doppler resolution in which the line width due to Doppler broadening (see Section 2.3.4) is reduced. This is achieved by observing the specttum in a direction perpendicular to that of the beam. The molecules in the beam have zero velocity in the direction of observation and the Doppler broadening is reduced substantially. Fluorescence excitation spectra can be obtained with sub-Doppler rotational line widths by directing the laser perpendicular to the beam. The Doppler broadening is not removed completely because both the laser beam and the supersonic beam are not quite parallel. 

In order to observe such high-resolution fluorescence excifafion spectra, the laser must have a very small line width. To achieve this a ring dye laser, a modification of the dye laser described in Section 9.2.10, is used a line width as small as 0.5 MFIz (1.5 x 10 cm ) can be obtained. 

Fluorescence excitation spectra of fairly large molecules in a supersonic jet are simplified, vibrationally, due to depopulation of low-lying vibrational levels in the ground electronic 

The two bands appear very different. Their rotational structure is quite symmetrical but that of aniline shows a pronounced gap near the band centre whereas that of aniline Ar shows a grouping of intense lines. The reason for the difference is that the band of aniline is a type B band of a prolate asymmetric rotor (see Section 6.2.4.4) whereas that of aniline Ar is a type C band of an oblate asymmetric rotor. The electronic transition moment in aniline itself is directed along the b axis which is in the plane of the benzene ring and perpendicular to the C—N axis (which is the a axis). In the aniline Ar molecule, the argon atom sits on the benzene ring, attracted by the n electrons. The fact that the argon atom is relatively heavy causes a rotation of the principal axes on inertia  

Some examples illustrate the various applications of the intracavity absorption technique. 

Although most experiments have so far been performed with dye lasers, the colour-center lasers or the newly developed vibronic solid-state lasers with broad spectral-gain profiles (Sect.5.7.3) are equally well suited for intracavity spectroscopy in the near infrared. An example is the spec- 

Modern photomultipliers reach quantum efficiencies of Jjpjj = 0.2. With carefully designed optics it is possible to achieve a collection factor 5 = 0.1. Using photon counting techniques and cooled multipliers (dark pulse rate 10 counts/s), counting rates of Opg = 100 counts/s are already sufficient to obtain a signal-to-noise ratio S/R 8 at integration times of Is. 

Inserting this figure for npg into (6.22) illustrates that with = 1 absorption rates of n = 5-10 /s can already be measured quantitatively. Assuming a laser power of 1 W at the wavelength A = 500 nm which corresponds to a photon flux of n = 3 10 /s, this implies that it is possible to detect a relative absorption of AI/I 10. When placing the absorbing probe inside the cavity where the laser power is q times larger (q 10 to 100, Sect.8.2.3), this impressive sensitivity may be even further enhanced. 

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Fluorescence energy transfer experiments, in which the energy transfer from the excited DNA bases to a fluorescent ligand is monitored by fluorescence excitation spectroscopy, has been used to analyze the binding of the bisquinolizinium species 35 to DNA <2004ARK219>. 

Fluorescence excitation spectroscopy is thus a powerful technique for obtaining molecular information about systems of cellular size. At present, the technique is restricted to single small objects because of the requirement of angular integration of the emitted fluorescence. As work progresses, similiar information will be obtainable from spectra taken at a particular angle with respect to the exciting beam. This will allow extension of the photoselection concept to suspensions of particles and perhaps to individual cells. 

Strube, J. and Stolz, P. 2000. Fluorescence excitation spectroscopy for the evaluation of seeds. In Alfbldi, T., Lockeretz, W. and Niggli, U. (eds) Proceedings of the 13th International IFOAM Scientific Conference. Hochschulverlag ETH, Zurich, pp. 306-309. 

The jet-cooled species can be studied using standard laser techniques (Figure 13-4a) like laser-induced fluorescence (LIF or fluorescence excitation spectroscopy) or R2PI, as well as using their sophisticated double-resonance variants, like UV/UV or IR/UV double resonance spectroscopy (Figure 13-4b and 4c), to investigate molecules or clusters existing under various isomeric forms... 

We can provide the following summary for the decay behavior of simple aliphatic aldehydes and ketones with little or no vibrational excitation energy on the Sp manifold under "isolated" molecule conditions at room temperature. A typical fluorescence decay time (tp) measured by a single-photon time-correlated lifetime apparatus (248) is 2-5 ns (42,101,102). A typical fluorescence quantum yield (ketones measured by fluorescence excitation spectroscopy is 10-, but the value is somewhat lower for aliphatic aldehydes (101,102). These values indicate that the radiative process (kp) is lO -lO s-1, three orders of magnitude slower than the total rate of nonradiative processes (kpjp) of 10 10 s-1. A typical radiative lifetime (tr) is 0.1 0.5 ps for aliphatic aldehydes and 0.1 ps for aliphatic ketones. 

For most carbonyl compounds, kp is approximately independent of vibrational excitation energy (Eypp,), whereas kfjR usually increases with Evpp,. Therefore, p becomes smaller and ip becomes shorter as Evpb increases. Typically, highly sensitive technique of fluorescence excitation spectroscopy permits measurement of Op over an extensive range of Evib- Hence the rates of radiative transitions as well as the rates of radiationless transitions of SVLs and SRVLs can be readily determined (135). For simple carbonyl compounds with small amounts of vibrational energy in the Sp state, collision-induced processes can become important at pressures above a few torr, since the lifetimes (tp) are comparable to the mean collision times (tu) at these pressures. Rate data reported for a number of aliphatic carbonyls are summarized in Table 2. 

A few years ago, a powerful version of molecular optical spectroscopy with supersonic beams and jets was developed by Smalley, Wharton and Levy . Supersonic expansion of molecules in an inert carrier gas yields an ideal spectroscopic sample. As a result of the expansion, the translational temperature of the carrier gas decreases to extremely low values (below O.I K). The flow is collisionless so that even extremely unstable species survive. Special attention was paid to fluorescence excitation spectroscopy but the technique is by no means limited to this type of spectroscopy. (Because of fundamental difficulties, however, direct measurement of light absorption in molecular beams is not easy.) Cooled molecules in the beam are electronically excited with a tunable dye laser. The emitted fluorescence is detected and plotted against the wavenumber of the exciting radiation. The obtained fluorescence excitation spectrum is generally very similar to the corresponding absorption spectrum. The technique was used for analysis of the spectra of interesting vdW molecules He. .. NOj, He... Ij, X. .. tetrazine and Xj. .. tetrazine (X = He, Ar, H ) complexes . 

Juzenas, P., Juzeniene, A., Kaalhus, O., Iani, V., and Moan, J. (2002a) Noninvasive fluorescence excitation spectroscopy during application of 5-aminolevulinic acid in vivo, Photochem. Photobiol. Sci., 1 745-748. 

Taken together, the carotenoid Sj and S, kinetic data give an overall quantum yield of energy transferor-80%, in qualitative agreement with that observed by steady state fluorescence excitation spectroscopy. 

Polymer rose bengal beads are most easily analyzed by fluorescence excitation spectroscopy. The emission spectra of immobilized dyes are presented as a function of solvent, of C-6 counter ion and dye-dye aggregation in this review. 

At this point it is relevant to note the terminology employed in this chapter the expression laser-induced fluorescence (LIF) is used as a general term describing any fluorescence that is excited using a laser. A fluorescence excitation spectrum shows fluorescence emission yield as a function of excitation wavelength that is, it is similar to an absorption spectrum when that absorption results in radiative emission. It is noted that some authors reserve LIF as a synonym for fluorescence excitation spectroscopy. Dispersed fluorescence refers to dispersion of the emitted fluorescence light into its component wavelengths, that is, production of an emission spectrum. 

Tao Tao, C., Dagdigian, P.J. Laser fluorescence excitation spectroscopy of the GcAr van der Waals complex, J. Chem. Phys. 118 (2003) 1242-1252. 

Fig. 1.23 Level scheme and experimental arrangement for fluorescence excitation spectroscopy...

Laser-induced fluorescence (LIF) has a large range of applications in spectroscopy. First, LIF serves as a sensitive monitor for the absorption of laser photons in fluorescence excitation spectroscopy (Sect. 1.3.1). In this case, the undispersed total fluorescence from the excited level is generally monitored (see Sect. 1.3.1). 

Often the absorption spectra of several pollutants overlap. It is therefore not possible to determine the specific concentrations of different pollutants from a single absorption measurement at a given wavelength k. Either several well-selected excitation wavelengths A/ have to be chosen (which is time consuming for in situ measurements) or time-resolved fluorescence excitation spectroscopy can be used. If the excited states of the different components have sufficiently different effective lifetimes, time-gated fluorescence detec-... 

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