Fluorescence life time distribution

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

The time distribution of the x-ray photons incident on the detector in an x-ray fluorescence spectrometer is described by Poisson statistics, providing that the half-life of the radioisotope excitation source is long compared to the data accumulation period, or providing that the x-ray tube output is truly constant. This means that both the x-ray tube current and voltage must be constant, with negligible drift or line frequency ripple. The arrival of photons at the detector is random in time as illustrated in Fig. 4.40. 

Photophysical properties and photochemical reactions of photoactive organic molecules in solid media may differ from those in homogenous solutions, in terms of the life time of the excited state, efficiency of radiationless quenching, diffusion of excited molecules, etc. [1, 2], Accordingly, the quantum yield of fluorescence, and the distribution and stereochemistry of the photochemical products can be greatly changed. Therefore, the stody of the photoprocesses of organic molecules in solid media is of interest because it could yield various applications such as solid dye laser, nonlinear optics, reaction media for controlled photochemical reactions and so on. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

The biodistribution of plasmid can be determined by measuring the rate of disappearance of radiolabeled DNA from the bloodstream and its accumulation in tissues or by the use of fluorescence microscopy to trace the leakage of dye-labeled plasmids from the vasculature. Pharmacokinetic analysis of in vivo disposition profiles of radiolabeled plasmid provides useful information on the overall distribution characteristics of systemically administered plasmids, with one critical limitation. The radiolabel represents both intact plasmid and its metabolites. The plasma half-life of plasmid is less than 10 min, and hence tissue distribution and pharmacokinetic parameters of plasmid calculated on the basis of total radioactivity are not valid at longer time points. Thus, polymerase chain reaction and Southern-blot analysis are required to establish the time at which the radiolabel is no longer an index of plasmid distribution. 

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