Stimulated fluorescence

A number of surface-sensitive spectroscopies rely only in part on photons. On the one hand, there are teclmiques where the sample is excited by electromagnetic radiation but where other particles ejected from the sample are used for the characterization of the surface (photons in electrons, ions or neutral atoms or moieties out). These include photoelectron spectroscopies (both x-ray- and UV-based) [89, 9Q and 91], photon stimulated desorption [92], and others. At the other end, a number of methods are based on a particles-in/photons-out set-up. These include inverse photoemission and ion- and electron-stimulated fluorescence [93, M]- All tirese teclmiques are discussed elsewhere in tliis encyclopaedia. 

To obtain the control and stimulated fluorescence histograms on the same scale, it is usually necessary to analyze the samples with fluorescence log amplification setting on the flow cytometer. 

Another technique that often utilises the UV spectral range is Fluorescence Spectroscopy, ft also relies on a UV excitation, and subsequent emission perpendicular to the incident beam (see Figure 7.9). The emission can either take place with the same frequency (resonance fluorescence) or at a lower frequency (stimulated fluorescence). The latter phenomenon is rooted in the ability of the UV excited state to interact with the local enviromnent, typically through the excitation of vibrational states of the surrounding part of the protein molecule or of the solvent molecules. 

Here (A) and crp (A) are the cross-sections for absorption and stimulated fluorescence at A, respectively, and mo is the population of the ground state. The first exponential term gives the attenuation due to reabsorption of the fluorescence by the long-wavelength tail of the absorption band. The attenuation becomes more important, the greater the overlap between the absorption and fluorescence bands. The cross-section for stimulated fluorescence is related to the Einstein coefficient by... 

Stimulated fluorescence appears with a delay of about 500 fs relative to the A [ absorption, although both absorption and fluorescence stem from the same state, proved by decay measurements with picosecond time-resolved spectroscopy. This delayed appearance of stimulated fluorescence is caused by a quickly increasing and short-lived absorption A0 located in nearly the same spectral range as the fluorescence. The authors assumed that the instant absorption A0 is caused by a state located slightly below the level reached by excitation with 4eV. This state 2,... 

In the case of 5T and 6T, the excited states with B symmetry near 4 eV can be occupied by allowed one-photon excitation from the A ground state. For 3T and 4T, states of A symmetry lie next to the excited state. They need vibronic coupling to be excited. The initially occurring absorption A0 is assumed to start from one of the higher electronic states near 4 eV. Relaxation processes from these states may be responsible for the decay of A0 during the first picosecond and for the delayed increase of stimulated fluorescence of 3T-6T. 

FIG. 2. Steady state absoption and stimulated fluorescence spectra of cresyl violet in acetonitrile (a), methanol (b), and ethanol (c) at room temperature. Exciting wavelength is indicated by the arrow. 

Normalized steady state absorption and stimulated fluorescence spectra of cresyl violet in polar solvents are shown in Fig. 2. Stimulated spectra were calculated from the fluorescence spectra divided by the square of the wavenumber. Exciting wavelengths in each solvent used for the measurements are also indicated in the figure by arrows. Mirror image relation between... 

FIG. 3. Estimation of the Sn -Sl absorption spectrum of cresyl violet in ethanol. 1 observed spectrum at 166ps 2 steady state absorption spectrum 3 stimulated fluorescence spectrum 4 superposition of absorption and stimulated fluorescence spectra 5 the Sn -Sl absoiption spectrum. 

For small particles, and in many other cases, one will use fluorescence instead of transmission. However, saturation effects such as we saw for transmission mode also occur in fluorescence as well (Troger et al. 1992 Castaner and Prieto 1997). The classic case here is that of a thick piece of pure metal such as Cu. In this material, the ratio of the resonant to non-resonant absorption is about 85 15. This means that for every 100 incident photons, 85 of them create -holes and thus could stimulate fluorescence. Now, suppose the resonant absorption goes up by 10% due to EXAFS. Now the ratio is 93.5 15, or about 86.1 13.9. Thus, the resonant process accounts for 86.1% of the total, which means the fluorescence intensity only goes up by 1.3% instead of 10%. This example shows that, again, the response saturates as a function of the absorption one wants to measure. 

Experimentally, the singlet states of free-base and zinc-porphyrins can be conveniently monitored not only by fluorescence spectroscopy but also by picosecond transient absorption (Fig. 24). By comparison with the absorp-tion/emission spectra of Fig. 22, it can be seen that the transient spectra consist of a broad featureless positive absorption throughout the visible region, with superimposed bleaching of the groimd-state Q-bands and additional apparent bleaching corresponding to stimulated fluorescent emission. As such. 

Hu, C. and Voss, K.J. (1998). Measurement of solar-stimulated fluorescence in natural waters. Limnol. Oceanogr., 43,1198-1206. 

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