Fluorescence theory fluorescent molecules

FCS and the utilization of fluorescence intensity as the fluctuating quantity was originally introduced in the 1970s with a series of papers presenting the theory and the first experimental realization of the technique [8-11]. In the first FCS experiments, the average number of fluorescent molecules in the observation volume was about 10,000. However, it was still possible to extract the motion of individual molecules from the large uncorrelated bulk fluorescence. In this particular respect, FCS is perhaps the first realization of fluorescence-based single-molecule spectroscopy. 

The signal-to-noise ratio in such a fluorescence fluctuation experiment is of the order [<<57/2>/<7/>2]1/2. This is obviously of order <<5iV2>1/2/ where < SN2y is the mean-square fluctuation in the number of fluorescent molecules in the illuminated volume and <7V> is the average number of molecules in this same volume. From fluctuation theory (cf. Section 5.5) it follows that this fraction is of order 1 /V or 1//Fc where c is the concentration of fluorescing molecules. Thus in order to study the concentration fluctuations it is necessary to either look at a very dilute solution or to focus the laser light so that V is very small. 

The choice of fluorescent probe depends on a variety of factors. It has already been pointed out that what is determined directly is information which characterises the distribution of orientations of the fluorescent molecules. The ideal experiment would be one in which the polymer molecules themselves contained fluorescent groups. Stein has considered the theory of the fluorescence method specifically for a uniaxially oriented fluorescent rubber but no experiments to study orientation have been reported for such a system. Nishijima et al have, however, made some qualitative observations on the polarisation of the fluorescent light from polyvinylchloride films which had been first stretched and then irradiated with light of wavelength 185 nm to produce fluorescent polyene segments. 

Fast concentration and sample injection are considered with the use of a theory of vibrational relaxation. A possibility to reduce a detection limit for trinitrotoluene to 10 g/cnf in less than 1 min is shown. Such a detection limit can by obtained using selective ionization combined with ion drift spectrometry. The time of detection in this case is 1- 3 s. A detection technique based on fluorescent reinforcing polymers, when the target molecules strongly quench fluorescence, holds much promise for developing fast detectors. 

Though theories have been proposed (32-35) to explain this phenomenon, the mechanism of fluorescence is still not yet fully understood. Jankow and Willis (36) proposed a mechanism which involves a direct excitation of the molecule or an impurity to an excited state, followed by internal conversion and then reversion back to the original state with emission of light. This mechanism can be explained as follows A molecule in the lowest vibrational level of the ground state A is transferred to a certain vibrational level in the excited state D. The molecule tends to cascade into the lowest vibrational level of state D by collisions with other excited molecules. It passes from state D to state C and then to state B by radiationless transi-... 

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

When the emissive state is a charge transfer state that is not attainable by direct excitation (e.g. which results from electron transfer in a donor-bridge-acceptor molecule see example at the end of the next section), the theories described above cannot be applied because the absorption spectrum of the charge transfer state is not known. Weller s theory for exciplexes is then more appropriate and only deals with the shift of the fluorescence spectrum, which is given by... 

Thus, at present, fluorescence spectroscopy is capable of providing direct information on molecular dynamics on the nanosecond time scale and can estimate the results of dynamics occurring beyond this range. The present-day multiparametric fluorescence experiment gives new opportunities for interpretation of these data and construction of improved dynamic models. A further development of the theory which would provide an improved description of the dynamics in quantitative terms with allowance for the structural inhomogeneity of protein molecules and the hierarchy of their internal motions is required. 

The theory of resonance transfer of electronic excitation energy between donor and acceptor molecules of suitable spectroscopic properties was first presented by Forster.(7) According to this theory, the rate constant for singlet energy transfer from an excited donor to a chromophore acceptor which may or may not be fluorescent is proportional to r 6, where r is the distance... 

This situation corresponds to the well-known saturation effect in the emission of most gas laser transitions, where, for the same reason, fewer upper-state molecules can contribute to the gain of the laser transition at the center of the doppler-broadened fluorescence line than nearby. When tuning the laser frequency across the doppler-line profile, the laser intensity therefore shows a dip at the centerfrequen-cy, called the Bennet hole or Lamb dip after W.R. Bennet who discovered and explained this phenomen, and W.E. Lamb 2) who predicted it in his general theory of a laser. 

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