• Title, Summary, Keyword: Fluorescence lifetime

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Specific Binding of Nile Red to Apomyoglobin

  • Chowdhury, Salina A.;Lim, Man-Ho
    • Journal of the Korean Chemical Society
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    • v.55 no.5
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    • pp.746-750
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    • 2011
  • Fluorescence correlation spectroscopy (FCS) is an emerging fluorescence technique used to study the dynamics of proteins on a millisecond to microsecond time scale at the single-molecule level. Solution pH-modulated protein conformational changes can be manifested by binding rate, fluorescence lifetime, and binding specificity of a probe molecule. The fluorescence lifetime of Nile red (NR) bound to apomyoglobin (apoMb) was measured to be $6{\pm}0.3$ ns, much longer than that in water solution ($2.9{\pm}0.2$ ns). As the unfolding population of apoMb increased by lowering pH of solution, the fraction for the longer lifetime of NR decreased with an increasing fraction for the shorter lifetime of NR in water. Unlike 1-anilino-8-naphthalene sulfonic acid, which has many lifetimes due to nonspecific binding to the unfolded apoMb, NR bound to apoMb possesses only a single lifetime. These results suggest that NR binds specifically to native apoMb and thus can be utilized to probe the folding/unfolding dynamics of apoMb using FCS.

Real-time Fluorescence Lifetime Imaging Microscopy Implementation by Analog Mean-Delay Method through Parallel Data Processing

  • Kim, Jayul;Ryu, Jiheun;Gweon, Daegab
    • Applied Microscopy
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    • v.46 no.1
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    • pp.6-13
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    • 2016
  • Fluorescence lifetime imaging microscopy (FLIM) has been considered an effective technique to investigate chemical properties of the specimens, especially of biological samples. Despite of this advantageous trait, researchers in this field have had difficulties applying FLIM to their systems because acquiring an image using FLIM consumes too much time. Although analog mean-delay (AMD) method was introduced to enhance the imaging speed of commonly used FLIM based on time-correlated single photon counting (TCSPC), a real-time image reconstruction using AMD method has not been implemented due to its data processing obstacles. In this paper, we introduce a real-time image restoration of AMD-FLIM through fast parallel data processing by using Threading Building Blocks (TBB; Intel) and octa-core processor (i7-5960x; Intel). Frame rate of 3.8 frames per second was achieved in $1,024{\times}1,024$ resolution with over 4 million lifetime determinations per second and measurement error within 10%. This image acquisition speed is 184 times faster than that of single-channel TCSPC and 9.2 times faster than that of 8-channel TCSPC (state-of-art photon counting rate of 80 million counts per second) with the same lifetime accuracy of 10% and the same pixel resolution.

Fluorescence Quenching of Green Fluorescent Protein during Denaturation by Guanidine

  • Jung, Ki-Chul;Park, Jae-Bok;Maeng, Pil-Jae;Kim, Hack-Jin
    • Bulletin of the Korean Chemical Society
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    • v.26 no.3
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    • pp.413-417
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    • 2005
  • Fluorescence of green fluorescent protein mutant, 2-5 GFP is observed during denaturation by guanidine. The fluorescence intensity decreases exponentially but the fluorescence lifetime does not change during denaturation. The fluorescence lifetime of the denatured protein is shorter than that of native form. As the protein structure is modified by guanidine, solvent water molecules penetrate into the protein barrel and protonate the chromophore to quench fluorescence. Most fluorescence quenchers do not affect the fluorescence of native form but accelerate the fluorescence intensity decay during denaturation. Based on the observations, a simple model is suggested for the structural change of the protein molecule during denaturation.

Effect of Ganglioside $G_{M3}$ on the Erythrocyte Glucose Transporter (GLUT1): Conformational Changes Measured by Steady-State and Time-Resolved Fluorescence Spectroscopy

  • Yoon, Hae-Jung;Lee, Min-Yung;Jhon, GiI-Ja
    • BMB Reports
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    • v.30 no.4
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    • pp.240-245
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    • 1997
  • Interactions between ganglioside $G_{M3}$ and glucose transporter, GLUT1 were studied by measuring the effect of $G_{M3}$ on steady-state and time-resolved fluorescence of purified GLUT1 in synthetic lipids and on the 3-O-methylglucose uptake by human erythrocytes. The intrinsic tryptophan fluorescence showed a GLUT 1 emission maximum of 335 nm, and increased in the presence of $G_{M3}$ by 12% without shifting the emission maximum, The fluorescence lifetimes of intrinsic tryptophan on GLUT1 consisted of a long component of 7.8 ns and a short component of 2,3 ns and $G_{M3}$ increased both lifetime components. Lifetime components were quenched by acrylamide and KI. Acrylarnide-mduced quenching of long-lifetime components was partly recovered by $G_{M3}$ However. KI-induccd quenching of short- and long-lifetime components was not rescued by $G_{M3}$. The anisotropy of 1.6-diphenyl-1.3.5-hexatriene (DPH)-probed dimyristoylphosphatidylcholine (DMPC) model membrane was also increased with $G_{M3}$ incorporation, The transport rate of 3-O-methylglucose increased by 20% with $G_{M3}$ incorporation on the erythrocytes, Therefore, $G_{M3}$ altered the environment of lipid membrane and induced the conformational change of GLUT1.

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Rapid Synthesis of AgInS2/ZnS Core/Shell Nanoparticles and Their Luminescence Property

  • Lee, Seung Jae;Kim, Da Hea;Jung, Jongjin;Park, Joung Kyu
    • Rapid Communication in Photoscience
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    • v.4 no.2
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    • pp.45-47
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    • 2015
  • We have successfully synthesized $AgInS_2$ core and $AgInS_2$/ZnS core/shell nanoparticles by the sonochemical method. The ultrasonic based $AgInS_2$ and $AgInS_2$/ZnS nanoparticle synthesis can be utilized as a simple and rapid method. The $AgInS_2$/ZnS nanoparticles show the higher fluorescence intensity and quantum yield than $AgInS_2$ nanoparticles. Fluorescence wavelength of $AgInS_2$/ZnS shows blue shift from 635 nm to 610 nm against $AgInS_2$ because of reducing the defect sites and increasing spatial confinements. For the fluorescence lifetime, $AgInS_2$/ZnS (124.8 ns) has longer lifetime than $AgInS_2$ (54.8 ns).

Excitation energy transfer from carotenoids probed by femtosecond time-resolved fluorescence spectroscopy

  • Akimoto, Seiji;Yamazaki, Iwao;Mimuro, Mamoru
    • Journal of Photoscience
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    • v.9 no.2
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    • pp.78-81
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    • 2002
  • Fluorescence rise and decay curves of carotenoids were measured in solutions and in pigment protein complexes with a femtosecond time-resolved fluorescence spectroscopy. For linear carotenoids, the S$_2$ lifetimes showed the maximum value around n = 9-10. The conjugation of a keto-carbonyl group shortened the S$_2$lifetime and prolonged the S$_1$lifetime. The excitation relaxation dynamics within carotenoids and the excitation energy transfer kinetics from carotenoids to chlorophylls are discussed as a function of molecular structure of carotenoids.

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Fluorescence Resonance Energy Transfer in Calf Thymus DNA from a Long-Lifetime Metal-Ligand Complex to Nile Blue

  • Kang, Jung-Sook;Lakowicz, Josepb R.
    • BMB Reports
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    • v.34 no.6
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    • pp.551-558
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    • 2001
  • We extended the measurable time scale of DNA dynamics to submicrosecond using a long-lifetime metal-ligand complex, $[Ru(phen)_2(dppz)]^{2+}$ (phen=1,10-phenanthroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuPD), which displays a mean lifetime near 350 ns. We partially characterized the fluorescence resonance energy transfer (FRET) in calf thymus DNA from RuPD to nile blue (NB) using frequency-domain fluorometry with a high-intensity, blue light-emitting diode (LED) as the modulated light source. There was a significant overlap of the emission spectrum of the donor RuPD with the absorption spectrum of the acceptor NB. The F$\ddot{o}$rster distance ($R_0$) that was calculated from the spectral overlap was $33.4\;{\AA}$. We observed dramatic decreases in the steady-state fluorescence intensities of RuPD when the NB concentration was increased. The intensity decays of RuPD were matched the closest by a triple exponential decay. The mean decay time of RuPD in the absence of the acceptor NB was 350.7 ns. In a concentration-dependent manner, RuPD showed rapid intensity decay times upon adding NB. The mean decay time decreased to 184.6 ns at $100\;{\mu}M$ NB. The FRET efficiency values that are calculated from the mean decay times increased from 0.107 at $20\;{\mu}M$ NB to 0.474 at $100\;{\mu}M$ NB concentration. The use of FRET with a long-lifetime metal-ligand complex donor is expected to offer the opportunity to increase the information about the structure and dynamics of nucleic acids.

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DNA Dynamics: a Fluorescence Resonance Energy Transfer Study Using a Long-Lifetime Metal-Ligand Complex

  • Kang, Jung-Sook;Lakowicz, Joseph-R.;Piszczek, Grzegorz
    • Archives of Pharmacal Research
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    • v.25 no.2
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    • pp.143-150
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    • 2002
  • Fluorescent probes bound to DNA typically display nanosecond decay times and reveal only nanosecond motions. We extend the time range of measurable DNA dynamics using $[Ru(pby)_2(dppz)]^{2+}$ (bpy=2.2'-bipyridine, dppz=dipyrido[3,2-a2',3'-c]phenazine) (RuBD) which displays a mean lifetime near 90 ns. To test the usefulness of RuBD as a probe for diffusive processes in calf thymus DNA, we compared the efficiencies of fluorescence resonance energy transfer (FRET) using three donors which display lifetimes near 5 ns for acridine orange (AO), 22 ns for ethidum bromide (EB) and 92 ns for RuBD, with nile blue (NB) as the acceptor. The F rster distances for AO-NB, EB-NB and RuBD-NB donor-acceptor pairs were 42.3, 52.3, and $30.6{\;}{\AA}$, respectively. All three donors showed dramatic decreases in fluorescence intensities and more rapid intensity decays with increasing NB concentrations. The intensity decays of AO and EB in the presence of varying concentrations of NB were satisfactorily described by the one-dimensional FRET model without diffusion (Blumen and Manz, 1979). In the case of the long-lifetime donor RuBD, the experimental phase and modulation somewhat deviated from the recovered values computed from this model. The recovered NB concentrations and FRET efficiencies from the model were slightly larger than the expected values, however, the recovered and expected values did not show a significant difference. Thus, it is suggested that the lifetime of RuBD is too short to measure diffusive processes in calf thymus DNA.

Dynamics of Supercoiled and Relaxed pTZ18U Plasmids Probed with a Long-Lifetime Metal-Ligand Complex

  • Kang, Jung-Sook;Abugo, Omoefe O.;Lakowicz, Joseph R.
    • BMB Reports
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    • v.35 no.4
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    • pp.389-394
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    • 2002
  • $[Ru(bpy)_2(dppz)]^2+$ (bpy=2,2'-bipyfidine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) (RuBD), a long-lifetime metal-ligand complex, displays favorable photophysical properties. These include long lifetime, polarized emission, but no significant fluorescence from the complex that is not bound to DNA. To show the usefulness of this luminophore (RuBD) for probing the bending and torsional dynamics of nucleic acids, its intensity and anisotropy decays when intercalated into supercoiled and relaxed pTZ18U plasmids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The mean lifetimes for the supercoiled plasmids (< $\tau$ >=148 ns) were somewhat shorter than those for the relaxed plasmids (< $\tau$ >=160 ns). This suggests that the relaxed plasmids were shielded more efficiently from water. The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled plasmids (288 ns) than for the relaxed plasmids (355 ns). The presence of two rotational correlation times suggests that RuBD reveals both the bending and torsional motions of the plasmids. These results indicate that RuBD can be useful for studying both the bending and torsional dynamics of mucleic acids.

A Novel Fluorescent Dipyrido[3,2-a:2',3'-c]phenazine (dppz) Derivative Prepared by Amide Bonding

  • Choi, Chang-Shik;Jeon, Ki-Seok;Lee, Ki-Hwan;Yoon, Min-Joong;Kwak, Min-Hee;Lee, Sang-Woo;Kim, In-Tae
    • Bulletin of the Korean Chemical Society
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    • v.27 no.10
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    • pp.1601-1603
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    • 2006
  • A novel fluorescent dipyrido[3,2-a:2',3'-c]phenazine (dppz) derivative, 7-(4-methoxybenzoylamino) dppz (1), was synthesized by amide connection to position 7 of the dppz ring. Its fluorescence quantum yield ($\Phi$ = 0.21 in dichloromethane) was as high as that of the conventional 7-amino-dppz (3), and its fluorescence lifetime was much shorter than that of 3.