Time-resolved fluorescence spectroscopy data analysis

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. 

From the analysis of the data in the LIPID AT database (41), more than 150 different methods and method modifications have been used to collect data related to the lipid phase transitions. Almost 90% of the data is accounted for by less than 10 methods. Differential scaiming calorimetry strongly dominates the field with two thirds of all phase transition records. From the other experimental techniques, various fluorescent methods account for 10% of the information records. X-ray diffraction, nuclear magnetic resonance (NMR), Raman spectroscopy, electron spin resonance (ESR), infrared (IR) spectroscopy, and polarizing microscopy each contribute to about or less than 2-3% of the phase transition data records in the database. Especially useful in gaining insight into the mechanism and kinetics of lipid phase transitions has been time-resolved synchrotron X-ray diffraction (62,78-81). 

Both the theoretical materials and the experimental data presented in this book dearly demonstrate the significant progress that has recently been made in the application of stilbenes in chemistry, photochemistry, photophysics, materials sdence, biochemistry, biomedicine, and clinical research. This progress resulted to a great extent from interdisciplinary cooperation. Advances in synthetic chemistry that provided researchers in these areas with a wide assortment of stilbenes paved the way for their multiple applications in basic and applied research. Modifications of traditional physical techniques and advanced methods such as nano-, pico-, femtosecond absorption, fluorescence and vibrational time-resolved spectroscopy, and theoretical approaches to the analysis of experimental data ensure profound photophysical and photochemical investigations in the area. Experts in biochemistry, biomedicine, and medicine effectively used natural and synthetic stilbenes in biochemical and preclinical studies and recenfly in clinical trials. 

A general discussion of the use of least-squares fitting in fluorescence measurements may be found in (28). The global analysis of fluorescence data is discussed in (29). Commercially available time-resolved fluorimeters are typically sold with data analysis software included. Available stand-alone packages include the Globals Unlimited suite, which is capable of analysing both time- and frequency-domain data, stopped-flow kinetics, etc. The Center for Fluorescence Spectroscopy at the University of Maryland (USA) also offers software for frequency- and time-domain fluorescence lifetime analysis. 

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