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Environment-sensing merocyanine dyes for live cell imaging applications.

ABSTRACT: Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical properties, including dye brightness, environmental responsiveness, and photostability, through the synthesis of a library of 25 merocyanine dyes, derived from combinatorial reaction of 5 donor and 5 acceptor heterocycles. Four of these dyes showed optimal properties for specific imaging applications and were subsequently prepared with reactive side chains and enhanced aqueous solubility using a one-pot synthetic method. The new dyes were then applied within a biosensor design for Cdc42 activation, where dye mero60 showed a remarkable 1470% increase in fluorescence intensity on binding activated Cdc42 in vitro. The dye-based biosensors were used to report activation of endogenous Cdc42 in living cells.

SUBMITTER: MacNevin CJ

PROVIDER: S-EPMC3667669 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Environment-sensing merocyanine dyes for live cell imaging applications.

MacNevin Christopher J CJ Gremyachinskiy Dmitriy D Hsu Chia-Wen CW Li Li L Rougie Marie M Davis Tamara T TT Hahn Klaus M KM

Bioconjugate chemistry 20130116 2

Fluorescent biosensors based on environmentally sensitive dyes enable visualization and quantification of endogenous protein activation within living cells. Merocyanine dyes are especially useful for live cell imaging applications, as they are extraordinarily bright, have long wavelengths of excitation and emission, and can exhibit readily detectable fluorescence changes in response to environment. We sought to systematically examine the effects of structural features on key photophysical proper ...[more]

PMID: 23297747

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Project description:Structural modifications of previously reported merocyanine dyes (Toutchkine, A.; Kraynov, V.; Hahn, K. J. Am. Chem. Soc. 2003, 125, 4132-4145) were found to greatly enhance the solvent dependence of their absorbance and fluorescence emission maxima. Density functional theory (DFT) calculations have been performed to understand the differences in optical properties between the new and previously synthesized dyes. Absorption and emission energies were calculated for several new dyes using DFT vertical self-consistent reaction field (VSCRF) methods. Geometries of ground and excited states were optimized with a conductor-like screening model (COSMO) and self-consistent field (SCF) methods. The new dyes have enhanced zwitterionic character in the ground state and much lower polarity in the excited state, as shown by the DFT-VSCRF calculations. Consistently, the position of the absorption bands are strongly blue-shifted in more polar solvent (methanol compared to benzene), as predicted by the DFT spectral calculations. Inclusion of explicit H-bonding solvent molecules within the quantum model further enhances the predicted shifts and is consistent with the observed spectral broadening. Smaller but significant spectral shifts in polar versus nonpolar solvent are predicted and observed for emission bands. The new dyes show large fluorescence quantum yields in polar hydrogen-bonding solvents; qualitatively, the longest bonds along the conjugated chain at the excited S1 state minimum are shorter in the more polar solvent, inhibiting photoisomerization. The loss of photostability of the dyes is a consequence of the reaction with and electron transfer to singlet oxygen, starting oxidative dye cleavage. The calculated vertical ionization potentials of three dyes I-SO, AI-SO(4), and AI-BA(4) in benzene and methanol are consistent with their relative photobleaching rates; the charge distributions along the conjugated chains for the three dyes are similarly predictive of higher reaction rates for AI-SO(4) and AI-BA(4) than for I-SO. Time-dependent DFT calculations were also performed on AI-BA(4); these were less accurate than the VSCRF method in predicting the absorption energy shift from benzene to methanol.

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Environment-sensing merocyanine dyes for live cell imaging applications.

Publications

Environment-sensing merocyanine dyes for live cell imaging applications.

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