Fluorescence Fluorescent light, polarization

Light absorption and fluorescence, FLUORESCENCE LIGHT POLARIZATION LIGHT SCATTERING... 

Donoi—acceptoi chromogens in solution are often strongly affected by the nature of the solvent or the resinous substrate in which they are dissolved. The more polar the solvent or resin, the longer the wavelength of the fluorescent light emitted. Progressing from less polar to more polar solvents, the bathochromic, or reddening, effect of the solvents on the dye increases in the order of aUphatics < aromatics < esters < alcohols < amides. 

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. 

Like Raman scattering, fluorescence spectroscopy involves a two-photon process so that it can be used to determine the second and the fourth rank order parameters. In this technique, a chromophore, either covalently linked to the polymer chain or a probe incorporated at small concentrations, absorbs incident light and emits fluorescence. If the incident electric field is linearly polarized in the e direction and the fluorescent light is collected through an analyzer in the es direction, the fluorescence intensity is given by... 

Because there is no phase relation between the light emitted by different molecules, fluorescence can be considered as the result of three independent sources of light polarized along three perpendicular axis Ox, Oy, Oz without any phase relation between them. Ix, Iy, Iz are the intensities of these sources, and the total intensity is I = Ix + Iy + Iz. The values of the intensity components depend on the polarization of the incident light and on the depolarization processes. Application of the Curie symmetry principle (an effect cannot be more dissymmetric than the... 

One can employ linearly polarized light to excite selectively those fluorophores that are in a particular orientation. The difference between excitation and emitted light polarization changes whenever fluorophores rotate during the period of time between excitation and emission. The magnitude of depolarization can be measured, and one can therefore deduce the fluorophore s rotational relaxation kinetics. Extrinsic fluorescence probes are especially useful here, because the proper choice of their fluorescence lifetime will greatly improve the measurement of rotational relaxation rates. One can also determine the freedom of motion of the probe relative to the rotational diffusion properties of the macromolecule to which it is attached. When held rigidly by the macromolecule, the depolarization of a probe s fluorescence is dominated by the the motion of the macromolecule. 

Anisotropy. Light emitted from excited molecules immediately after absorption is always partially polarized, whether or not the exciting beam consists of plane polarized light. When light polarized in a vertical plane is used for excitation, part of the emitted light (of intensity lv) will have its electric vector parallel to that of the exciting light. The remainder of intensity /, will be polarized in a horizontal plane. The polarization P of the emitted radiation is defined by Eq. 23-19 and the anisotropy R by Eq. 23-20. After excitation by a laser pulse both the fluorescence and its anisotropy decay with time and can be measured. The decay of R (but not of P) can usually be described as the sum... 

Figure 2.4 (Upper) White-light (polarized) photomicrograph, in reflected mode, of an suspension with a significant emulsified oil content. With polarized light, the clays (C) appear bright, but the oil droplets cannot be seen at all. (Lower) In this reflected-light photomicrograph, of the same field of view as above, the fluorescence mode shows bright oil droplets in a dark water-continuous phase. In this photograph the clays cannot be seen. From Mikula [66], Copyright 1992, American Chemical Society.

The polymer absorption is intense ap 555 7.5 x 105 cm-1 for light polarized parallel to the PA chains and the dichroic ratio is 25. The polymer is not fluorescent. 

The maximum absorption per chain for light polarized parallel to the PDA chains is at least as intense as for PA. The measured dichroic ratio is about 120 [117,118]. It may be smaller in other PDAs for instance, about 25 in pTS [123]. As for trans-PA, solid PDAs are not fluorescent (there is a small solution fluorescence). 

The absorption of light is proportional to the scalar product of the incident electric field and of a molecular vector named the transition moment. Thus, excitation of an isotropic population of fluorescent species by polarized light generally creates a temporary anisotropic population of excited molecules. Molecular motions progressively destroy this anisotropy, and affect the polarization of the reemitted fluorescence light which can be studied using pulse fluorometry techniques. 

The fluorescence depolarization technique for mobility and ordering is based on the fact that the probability of absorption and emission is directional. Light polarized along a certain axis will preferably excite molecules oriented with their transition dipole moment in the same direction. The probability varies with cos 0, where 0 is the angle between the transition dipole moment and the electric field vector of the light. Emission of a photon obeys the same cos 0 (28) rule. That means that a molecule oriented with its transition dipole moment along the Z-axis will be likely to emit a photon with the same polarization. In the depolarization technique, polarizers are used to quantify the intensity of the parallel (ly) and perpendicular (Ij.) components to the original direction of polarization. 

The intensity of the fluorescence at time t, polarized at a partfcular an e to the excitation polarization, is proportional to the second power of the component of the emission dipole in the same direction and the probability P(t) that the sample is still excited at time t. For an individual transition dipole, the intersity of light polarized parallel and perpendicular to the exciting light (J (t) and Ji(t)) at time t will be given by... 

Optical Microscopy. Optical microscopy involves the use of transmitted light, reflected light, polarized light, fluorescence, and more recently, techniques such as confocal microscopy. Each of these variations has particular strengths and applicability. 

One commercial system is based on fluorescence polarization. Molecules absorbing polarized light emit polarized fluorescent light. The de-... 

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