Novel Fluorophores for Single-Molecule Imaging

Willets, Katherine A.; Ostroverkhova, Oksana; He, Meng; Twieg, Robert J.; Moerner, W. E.

doi:10.1021/ja029100q

Cited by 97 publications

(77 citation statements)

References 38 publications

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“…However, the disadvantages of the azido DCDHF system are: it is not genetically targeted, as fluorescent proteins are; DCDHFs with a primary amine exhibit more blinking than those with secondary or tertiary amines (see Figure 7 and reference [13]); and the photoactivation is irreversible (once the fluorophore photobleaches, it cannot be reactivated, which is possible in some photoswitches[55-60]).…”

Section: Structure-property Designmentioning

confidence: 99%

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

et al. 2009

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There is a persistent need for small-molecule fluorescent labels optimized for single-molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red-shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed "DCDHF" fluorophores, for use in live-cell imaging based on the push-pull design: an amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a π-rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small-molecule fluorophores, which are needed for super-resolution imaging schemes that require active control (here turning-on) of singlemolecule emission.

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Section: Structure-property Designmentioning

confidence: 99%

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

et al. 2009

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“…[1,2] Our purpose in this work was to design and characterize dyes that 1) respond to medium polarity by solvatochromic shifts of their fluorescence emission band, and 2) meet all the stringent requirements for single-molecule spectroscopy. [3,4] We have found that the amino-substituted perylene imides 5PI and 6PI (Scheme 1) indeed combine excellent detectability at the single-molecule level with substantial solvatochromic sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Paradoxical Solvent Effects on the Absorption and Emission Spectra of Amino‐Substituted Perylene Monoimides

Zoon

Brouwer

2005

ChemPhysChem

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In N-(2,5-di-tert-butylphenyl)-9-pyrrolidinoperylene-3,4-dicarboximide (5PI) the absorption and emission spectra display large solvatochromic shifts, but, remarkably, the Stokes shift is practically independent of solvent polarity. This unique behavior is caused by the extraordinarily large ground-state dipole moment of 5PI, which further increases upon increasing the solvent polarity, whereas the excited-state dipole moment is less solvent dependent. In the corresponding piperidine compound, 6PI, this effect is much less important owing to the weaker coupling between the amino group and the aromatic imide moiety, and in the corresponding naphthalimide, 5NI, it is absent. The latter shows the conventional solvatochromic behavior of a push-pull substituted conjugated system, that is, minor shifts in absorption and a larger change in the emission energy with solvent polarity.

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“…The DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. [4][5][6][7][8][9][10][11][12][13][14] We have now reengineered the DCDHFs to make them photoactivatable: by replacing the amine with an azide, the fluorescence is quenched; upon photocleaving the azide, the amino fluorophore is regenerated, and the fluorescence returns. 13 In this paper, we review the properties of the DCDHF class in general, and then present the scheme that has been utilized to make them photoswitchable.…”

Section: Introductionmentioning

confidence: 98%

Photoactivatable DCDHF fluorophores for single-molecule imaging

et al. 2009

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We have designed and studied the photophysics of a class of organic fluorophores termed "DCDHFs," which were originally used as push-pull chromophores for nonlinear optical applications. In this paper, we describe the general photophysics of many realizations of the DCDHF class of single-molecule emitters. Moreover, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the singlemolecule level in living cells. This molecule and its relatives will provide a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single-molecule emission.

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Novel Fluorophores for Single-Molecule Imaging

Cited by 97 publications

References 38 publications

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

DCDHF Fluorophores for Single‐Molecule Imaging in Cells

Paradoxical Solvent Effects on the Absorption and Emission Spectra of Amino‐Substituted Perylene Monoimides

Photoactivatable DCDHF fluorophores for single-molecule imaging

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