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The majority of biological assays measure fluorescence intensity in order to ascertain the number of fluorophore labeled probes that bind selectively to the molecules of interest in the assay. The development of fluorescence intensity standards will make it possible to compare measurements between different laboratories and instruments. In addition to the standard materials, it is also necessary to develop measurement models to facilitate the proper use of the fluorescence intensity standards. A research program in fluorescence spectroscopy supports the development of fluorescence intensity standards and models. A major effort is underway to study the behavior of fluorophore labeled single strand DNA (ssDNA) hybridized to complementary ssDNA. The quenching of the label fluorescence by nucleotides and other nearby fluorophores is a major concern in quantitation of DNA microarray assays and real time polymerase chain reactions(PCR). Work is underway to study the fluorescence properties of phycoerythrin(PE) proteins. PE are comprised of an array of fluorophores and their photophysical behavior is more complicated than that of a single fluorophore such as fluorescein. The fluorescence properties of fluorophores change whenever they are immobilized on synthetic microspheres or modified glass surfaces. In addition to spectral shifts and quantum yield changes, there are also changes in absorbance and emission due to orientation of molecular dipole moments. It is important to understand these changes in order to manipulate the spectral properties of prospective standards. Another important effect in quantitative fluorescence measurements is the photodegradation of the fluorophore. Most analytical instruments(e.g. DNA microarrays) utilize intense laser beams so that photodegradation is significant. Furthermore the photodegradation properties may change upon immobilization to a surface. The research work on surface fluorescence is supported by other spectroscopic surface probes such as surface plasmon resonance (SPR) and surface enhanced Raman scattering (SERS).. | ||
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