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Principles Of Molecular Photochemistry: An

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Principles Of Molecular Photochemistry: An

fluorescence resonance energy transfer, which was originally observed between the two forms of fluorescein (h- and l-isomers), is now a well-established phenomenon that has been widely exploited in biological applications. this phenomenon is usually explained in terms of the fluorescein isothiocyanate (fitc) molecule being a suitable fluorophore for labeling cellular structures with which it has a significant absorption cross-section, and which are also rich in thiol groups, such as proteins, nucleic acids, polysaccharides and glycoproteins. as a result, transfer of excitation energy from the dye to the target may lead to the accumulation of energy in the form of long-lived triplet state that subsequently decays to yield emission (fret excitation and emissionsection 4.4 ). several other fluorophores, including alexa fluor dyes, have been successfully applied to image specific interactions in living cells by fret. the principle can also be used with synthetic dye-protein conjugates, in which the emission spectrum of the donor is broadened by the intermolecular dipole-dipole interaction with the acceptor, but the overall fluorescence quantum yield remains unchanged (fret sample preparationsection 10.2).

in cases where a photobleaching-free imaging system is essential for long-term studies, the most logical approach is to select a fluorophore that has a large extinction coefficient. this ensures that the desired emission intensity will be obtained from a low excitation intensity, and consequently the fluorophore will be less susceptible to photobleaching. it is also necessary to minimize other perturbations of the fluorophore's conformation that might affect the quantum yield, such as nonradiative energy transfer or molecular collisions. this is achieved by using a fluorophore in which the excited-state lifetime is relatively long, as is the case with some fluorescein isothiocyanate (fitc) derivatives (fluorescence microscopy accessories and reference standardssection 23.1) and fluorescein (fluorescein section). while it is difficult to predict the photostability of a particular fluorophore, the best approach is to select a fluorophore with a proven track record for photostability. for example, the use of fluorescein allows the simultaneous evaluation of the fluorescence-quenching effect of the intracellular environment. the use of rhodamine dyes, such as rhodamine 123 or other rhodamine derivatives, allows the evaluation of plasma membrane integrity and intracellular ph. green fluorescent proteins (gfp) also provide a reliable means of expressing membrane-permeant protein labels in cells; gfps are relatively stable because their amino acid sequence has been optimized to improve the photostability of the protein. 3d9ccd7d82


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