Spectral Identification of Specific Photophysics of Cy5 by Means of Ensemble and Single Molecule Measurements

Huang, Zhengxi; Ji, Dongmei; Wang, Sufan; Xia, Andong; Koberling, Felix; Patting, Matthias; Erdmann, Rainer

doi:10.1021/jp0562936

Cited by 74 publications

(127 citation statements)

References 56 publications

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“…Using the known Cy5 triplet lifetime (~3 μsec),39 however, does not yield significant enhancements at these excitation intensities. Further, in contrast to most single molecule experiments, the steady-state triplet population in our fully aerated solutions is much lower than that of the longer-lived spectrally-shifted cis-Cy5 isomer 37. The strong agreement between the model and the experimental data demonstrates that the long dark state residence greatly increases the probability of simultaneous excited state population, thereby enhancing Cy3 emission at very low secondary excitation intensities via FRET inhibition.…”

Section: Resultsmentioning

confidence: 50%

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FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

et al. 2010

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Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally-imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally-shifted, dark state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement vs. modulation frequency directly yields dark state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield ≫10-fold sensitivity improvements through Synchronously Amplified Fluorescence Image Recovery (SAFIRe).

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Section: Resultsmentioning

confidence: 50%

“…4A). 37 Changing other experimental parameters (e.g. dark state quantum yield, excitation rates) only shifts the enhancement curve, without affecting dynamic range.…”

Section: Resultsmentioning

confidence: 99%

FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

et al. 2010

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“…Using the same argument, the efficiency of transcription site detection by F2 is 55% 6 5%. As probe labeling efficiency was estimated spectrophotometrically to be near 100%, we suggest that some probe molecules may be nondetectable because the fluorophore is in a dark state (Widengren and Schwille 2000;Huang et al 2006), a phenomenon that affects Cy5 (F2) more than Cy3 (F1).…”

Section: Single-molecule Pol II Transcription Detectionmentioning

confidence: 92%

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Revyakin¹,

Zhang²,

Coleman³

et al. 2012

Genes Dev.

109

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Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at singlemolecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

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“…22 The photoinduced isomerization and back-isomerization of Cy5 have been characterized by Widengren et al using fluorescence correlation spectroscopy, 23 and more recently, Huang et al characterized the spectroscopic behavior of this dye by means of ensemble and single-molecule measurements. 24 However, despite its widespread popularity, the photophysics of Cy3 has not been investigated thoroughly to date. It has been well-established that carbocyanines with short polymethine chains (such as Cy3) photoisomerize from the first excited state more efficiently than the longer analogues (such as Cy5).…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Properties and Photophysics of the Sulfoindocyanine Cy3 Linked Covalently to DNA

et al. 2007

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The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5' ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5' dsDNA). Substantial differences were also observed between the 5'-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5' ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5' ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5' phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.

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Spectral Identification of Specific Photophysics of Cy5 by Means of Ensemble and Single Molecule Measurements

Cited by 74 publications

References 56 publications

FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

Transcription initiation by human RNA polymerase II visualized at single-molecule resolution

Fluorescence Properties and Photophysics of the Sulfoindocyanine Cy3 Linked Covalently to DNA

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