Instrumentation polarization decay

Calibration. In general, standards used for instrument calibration are physical devices (standard lamps, flow meters, etc.) or pure chemical compounds in solution (solid or liquid), although some combined forms could be used (e.g., Tb + Eu in glass for wavelength calibration). Calibrated lnstr iment parameters include wavelength accuracy, detection-system spectral responsivity (to determine corrected excitation and emission spectra), and stability, among others. Fluorescence data such as corrected excitation and emission spectra, quantum yields, decay times, and polarization that are to be compared among laboratories are dependent on these calibrations. The Instrument and fluorescence parameters and various standards, reviewed recently (1,2,11), are discussed briefly below. 

The first photoelectric fhiorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5], In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33], The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fhiorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35]. 

As reviewed in the introduction, the luminescence from polymers and biopolymers may be described in terms of spectral shape, quantum yield of emission, decay time characteristics and polarization properties. The recent rapid increase in interest in the usefulness of luminescence techniques to study the structure and prtqwrties of molecular systems is partly due to the now ready availability of reliable instrumentation. Although the apparatus necessary for studying the spectral characteristics of luminescence is well established and has been r iewed in detail by several authors there have been recent rapid developments in the techniques available for time-... 

Thus after a period of tf the intensity has dropped to 37% of h, that is 63% of the molecules return to the ground state before tf. In many cases the above expression needs to be modified into more complex expressions. First of all it is assumed that the instrument yields an infinite (or very) short light pulse at time zero. In cases where tf is small Ig must be replaced by a function, which describes the lamp profile of the instrument. Also, more than one lifetime parameter is often needed to describe the decay profile, which is l(t) must be expressed as a sum of exponentials. Finally the concept of anisotropy should be mentioned. Anisotropy is based on selectively exciting molecules with their absorption transition moments aligned parallel to the electric vector of polarized light. By looking at the polarization of the emission the orientation of the fluorophore can be measured. The anisotropy of the system is defined as (Equation 6) (Rendell, 1987 Lakowicz, 2006) ... 

With respect to fiber-optic-based fluorescence sensing, most of the past approaches have employed either excitation or emission wavelength selectivity (10,11). The other selectivity parameters fluorescence lifetime, steady-state polarization, and rotational diffusion rates have received little if any attention. The acquisition of fluorescence lifetime information via optical fibers has been demonstrated previously (12) however, the time resolution and ability to resolve multiexponential decays of fluorescence were neither demonstrated nor possible with this earlier instrument. Recently, our own group (13,1 ) has developed and described the first fiber-optic-based fluorescence lifetime instrumentation capable of unequivocally determining single, double, and/or triple exponential decays of fluorescence in remotely-located samples. 

Anisotropic rotati onal diffusion has been more Nct sively studied using FD methods. In fact, the earliest reports on the anisotropic rotation of fluorophores concerned experiments performed using fixed-frequency phase-modulation fluorometers. At that time the phase-modulation instruments operated at only one or two fixed hrequendes. Hence, it was not possible to recover the anisotropy decay law. The experiments were performed by measuring the differential polarized phase angles as the temperature was varied. It is relatively simple to predict the maximum value of Ao) for known values of the lifetime and fundamental anisotropy. For an isotropic rotor, the predicted value of Aw is given by... 

Stiibiger and colleagues [36] used a similar approach (in addition to the MS analysis of lipid species subsequent to reelution from the TLC plate) to screen the compositions of various neutral (e.g., triacylglycerols) and polar (e.g., GPL and glyc-erosphingolipids [GSL]) lipid classes derived from crude lipid extracts of human plasma as well as soybean lecithin. These authors [36] have also provided evidence that combining TLC/MS with post source decay (PSD) MALDl MS helps to address structural problems such as the detailed fatty acyl composition and the differentiation of positional isomers sn- vs. sn- I). PSD may be considered an MS/MS-Uke experiment that can be performed on all TOP instruments equipped with a reflectron [20] but without the need of a dedicated collision ceU. About 70 different lipid species could be detected in just 50 jL of human blood plasma. 

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