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.
| Thivierge, Cliferson; Han, Junyan; Jenkins, Roxanne M et al. (2011) Fluorescent proton sensors based on energy transfer. J Org Chem 76:5219-28 |
| Jose, Jiney; Loudet, Aurore; Ueno, Yuichiro et al. (2011) Energy transfer cassettes in silica nanoparticles target intracellular organelles. Org Biomol Chem 9:3871-7 |
| Ueno, Yuichiro; Jose, Jiney; Loudet, Aurore et al. (2011) Encapsulated energy-transfer cassettes with extremely well resolved fluorescent outputs. J Am Chem Soc 133:51-5 |
| Jose, Jiney; Loudet, Aurore; Ueno, Yuichiro et al. (2010) Intracellular imaging of organelles with new water-soluble benzophenoxazine dyes. Org Biomol Chem 8:2052-9 |
| Han, Junyan; Gonzalez, Oswaldo; Aguilar-Aguilar, Angelica et al. (2009) 3- and 5-Functionalized BODIPYs via the Liebeskind-Srogl reaction. Org Biomol Chem 7:34-6 |
| Han, Junyan; Loudet, Aurore; Barhoumi, Rola et al. (2009) A ratiometric pH reporter for imaging protein-dye conjugates in living cells. J Am Chem Soc 131:1642-3 |
| Cieýýlik-Boczula, Katarzyna; Burgess, Kevin; Li, Lingling et al. (2009) Photophysics and stability of cyano-substituted boradiazaindacene dyes. Photochem Photobiol Sci 8:1006-15 |
| Jose, Jiney; Ueno, Yuichiro; Burgess, Kevin (2009) Water-soluble Nile Blue derivatives: syntheses and photophysical properties. Chemistry 15:418-23 |
| Wu, Liangxing; Loudet, Aurore; Barhoumi, Rola et al. (2009) Fluorescent cassettes for monitoring three-component interactions in vitro and in living cells. J Am Chem Soc 131:9156-7 |
| Jose, Jiney; Ueno, Yuichiro; Castro, Juan C et al. (2009) Energy transfer dyads based on Nile Red. Tetrahedron Lett 50:6442-6445 |
Showing the most recent 10 out of 35 publications
