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Comparative Study
. 2005 Mar 4;328(1):78-84.
doi: 10.1016/j.bbrc.2004.12.148.

Two-photon Induced Fluorescence of Cy5-DNA in Buffer Solution and on Silver Island Films

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Comparative Study

Two-photon Induced Fluorescence of Cy5-DNA in Buffer Solution and on Silver Island Films

Joanna Lukomska et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

We report the observation of a strong two-photon induced fluorescence emission of Cy5-DNA within the tunable range of a Ti:Sapphire laser. The estimated two-photon cross-section for Cy5-DNA of 400GM is about 3.5-fold higher than it was reported for rhodamine B. The fundamental anisotropies of Cy5-DNA are close to the theoretical limits of 2/5 and 4/7 for one- and two-photon excitation, respectively. We also observed an enhanced two-photon induced fluorescence (TPIF) of Cy5-DNA deposited on silver island films (SIFs). In the presence of SIFs, the TPIF is about 100-fold brighter. The brightness increase of Cy5-DNA TPIF near SIFs is mostly due to enhanced local field.

Figures

Fig. 1.
Fig. 1.
Emission spectra of two-photon induced fluorescence of Cy3-DNA and Cy5-DNA in hybridization buffer (5 mM Hepes, pH 7.5,0.1 M KCl, and 0.25 mM EDTA) and RhB in methanol. The concentrations of all three fluorophores were the same, 1.3 μM. The experimental conditions were kept constant during the measurements. The excitation was from femtosecond, tunable Ti:Sapphire laser.
Fig. 2.
Fig. 2.
The two-photon cross-sections for Cy3-DNA (⋯) and Cy5-DNA (—) in hybridization buffer calculated using RhB in methanol (- - -) as a reference [36]. The upper panel shows the one-photon absorption spectrum of Cy5-DNA. The horizontal bar marks the energy level of observed two-photon transition. This transition is not seen in the one-photon spectrum.
Fig. 3.
Fig. 3.
Frequency-domain intensity decays of Cy5-DNA in hybridization buffer with one-photon (top) and two-photon (bottom) excitation. The lifetimes are similar for one- and two-photon excitation, indicating that emission occurs from the same fluorescent state.
Fig. 4.
Fig. 4.
Emission anisotropy spectra for Cy5-DNA in buffer solution obtained with one- and two-photon excitation. The high anisotropy values indicate a low mobility of Cy5-DNA molecules. The ratio of two-photon to one-photon anisotropies is very close to 10/7, the theoretical value for parallel transitions.
Fig. 5.
Fig. 5.
The excitation anisotropy spectra of Cy5-DNA in buffer solution at 20°C and in 90% glycerol at −5°C. The anisotropies in glycerol are close to the maximal fundamental values 0.4 for one-photon and 0.57 for two-photon excitation. There is a weak one-photon transition around 430 nm with low anisotropy.
Fig. 6.
Fig. 6.
Two-photon-induced emission spectra of Cy5-DNA on SIFs and quartz. The brightness of Cy5-DNA on SIFs is approximately 100-fold higher than on quartz. In the top panel is shown the schematic of Cy5-dye deposited on SIFs.
Fig. 7.
Fig. 7.
The excitation power dependence of two-photon induced fluorescence of Cy5-DNA on SIFs. The excitation was 785 nm and emission was observed at 665 nm.
Fig. 8.
Fig. 8.
Frequency-domain intensity decays of Cy5-DNA on SIFs observed with one-photon (top) and two-photon (bottom) excitation. The lifetimes are significantly shorter than observed in solution or on quartz (Table 1).
Scheme 1.
Scheme 1.
Structures of Cy3-DNA and Cy5-DNA.

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