Polarized fluorescence measurements

Altkorn R and Zare R N 1984 Effects of saturation on laser-induced fluorescence measurements of population and polarization Annual Review of Physical Chemistry ed B S Rabinovitch, J M Schurr and H L Strauss (Palo Alto, CA Annual Reviews)... 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

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. 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Pitfalls in steady-state fluorescence measurements inner filter effects and polarization effects... 

The case of several populations of fluorophores having their own fluorescence decay i (t) and time constants characterizing r (t) deserves particular attention. In Section 5.3, it was concluded that an apparent or a technical emission anisotropy r(t) can be obtained by considering that the measured polarized components, I(t) and I (t), are the sums of the individual components (i.e. of each population) and by using Eq. (6.43). Hence... 

FLUORESCENCE MEASUREMENTS OF LIGAND BINDING. In principle, ligand binding may either enhance or quench the intrinsic or extrinsic fluorescence of its macromolecular receptor or it may change the polarization of the fluorescence emission (see below). 

One should be careful in measuring polarization, since if the sample is not fluorescing too much, errors in reading the intensities could be frequent. Also, depending on the instrument the student is using to perform the measurements, experiments could be easy or very hard to conduct. Therefore, it is important that all students perform each experiment described here so that they can learn how to conduct all experiments, difficult and easy, and also compare all the data obtained together. 

Fig. 18. Schematic of apparatus used to measure fluorescence kinetics with a streak camera. The Nd glass laser emits a train of one hundred 1.06 pm pulses separated by 6 ns. A single pulse in the earlier portion of the train is selected by a Pockels cell and crossed polarizers (Pi and P2). The high voltage pulse ( 5 ns) at the Pockels cell is supplied by a laser triggered spark gap and a charged line. The single pulse ( 8 ps, 109 W) can be amplified. The second harmonic is generated from a phase matched KDP crystal. Beam splitters provide two side beams beam (1) triggers the streak camera beam (2) arriving at the streak camera at an earlier time acts as a calibrating pulse. The main 0.53 pm beam excites the sample for fluorescence measurement. The fluorescence collected with f/1.25 optics is focused into the 30 pm slit of the streak camera. The streak produced at the phosphorescent screen is recorded by an optical multichannel analyzer. (After ref. 67.)...

Infrared dichroism is one of numerous methods used to characterize molecular orientation. The degree of anisotropy of the strained pol3rmers may also be accurately characterized by other techniques such as X-ray diffraction, birefringence, sonic modulus, polarized fluorescence and polarized Raman spectroscopy [2]. These techniques directly probe the orientational behavior of macromolecular chains at a molecular level, in contrast to the macroscopic information provided by mechanical measurements. 

Hi) UV/visible spectroscopy. In comparison with IR and fluorescence spectroscopic techniques, UV/visible spectroscopy is only occasionally used for characterizing monolayers. It can be applied if the monolayer contains molecules with 7i-electron systems of which the electron transitions are in the UV/visible part of the spectrum. By measuring polarized transmission spectra or reflection-absorption spectra at different angles of incidence, the second order parameter of the absorption transition dipole moment in the chromophoric groups caii be determined. In the case of a reflection-absorption configuration, the underlying theory is similar to that of IRRAS, i.e. based upon calculation of the reflection and transmission coefficients in a stratified-layer system and extended to account for the anisotropic nature of monolayers ). 

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