Dyes for intracellular imaging are underdeveloped relative to the sophisticated instrumentation available for observing fluorescence in cells. Superior probes would have several important characteristics. They should be small, noncytotoxic, water-soluble entities that do not interfere with the function of the molecules to which they are conjugated. Their spectroscopic properties should be such that they absorb very strongly at wavelengths that correspond to the excitation source used, then emit with high quantum yields. If several biomolecules are to be labeled and observed simultaneously (ie multiplexing), then sets of dyes are required that absorb strongly at one convenient excitation wavelength, but emit sharp intense fluorescence signals with different Stoke's shifts giving high resolutions. Modifications should be available to facilitate absorption at excitation wavelengths/powers that avoid autofluorescence from cellular biomolecules and cell damage. The emission wavelengths should occur at relatively long wavelengths (eg 800 nm) where cells are essentially transparent enabling easier detection. For single molecule detection methods, the probes should resist photodecomposition and """"""""blinking"""""""" involving non-fluorescent excited states. Finally, there are other properties that might be engineered into fluorescent probes. These include isotropy of transition dipoles to remove uncertainties caused by orientation factors in FRET- based distance measurements, and favorable lifetime characteristics for observation via gated measurements to increase signal-to-noise. In preliminary studies, a set of novel probes was devised for multiplexing in DNA sequencing. The conceptual basis for these is also applicable to intracellular imaging. They contain donor parts that absorb strongly at one excitation wavelength, twisted, but otherwise conjugated, linker systems that facilitate rapid """"""""through-bond energy transfer"""""""", and acceptor parts that emit at wavelengths far from the excitation source. Thus they can be excited with high cross-sections by a single laser, then fluoresce strongly at different, resolvable wavelengths. This proposal is to create similar sets of """"""""through-bond energy transfer cassettes"""""""" that are optimized for intracellular imaging, ie that conform to most or all of the criteria described above. They will be prepared (Burgess), optimized for superior spectroscopic properties (Burgess/Hochstrasser), and attached to a set of four model proteins (Schroeder/Burgess). The labeled-proteins will be transported into cells and observed using multicomponent, equilibrium fluorescence measurements to detect protein-protein interactions via FRET donor (Schroeder). The data collected will be analyzed via novel computer algorithms (Liu), then compared with that obtained by Hochstrasser et al for the same intracellular events observed at the single molecule level. This work proposed will provide superior intracellular probes for equilibrium and single molecule detection methods.
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