• Journal of the Korean Chemical Society
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• v.67 no.1
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• pp.64-67
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• 2023

• Bulletin of the Korean Chemical Society
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• v.32 no.2
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• pp.583-589
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• 2011

Fluorescence correlation spectroscopy (FCS) is a sophisticated and an accurate analytical technique used to study the diffusion of molecules in a solution at the single-molecule level. FCS is strongly affected by many factors such as the stability of the excitation power, photochemical processes, mismatch between the refractive indices, and variations in the cover glass thickness. We have studied FCS near the surface of a cover glass by using rhodamine 123 as a fluorescent probe and have observed that the surface has a strong influence on the measurements. The temporal autocorrelation of FCS decays with two characteristic times when the confocal detection volume is positioned near the surface of the cover glass. As the position of the detection volume is moved away from the surface, the FCS autocorrelation becomes one-component decaying; the characteristic time of the decay is the same as the faster-decaying component in the FCS autocorrelation near the surface. This observation suggests that the faster component can be attributed to the free diffusion of the probe molecules in the solution, while the slow component has its origin from the interaction between the probe molecules and the surface. We have characterized the surface contribution to the FCS measurements near the surface by changing the position of the detection volume relative to the surface. The influence of the surface on the diffusion of the probe molecules was monitored by changing the chemical properties of the surface. The surface contribution to the temporal autocorrelation of the FCS strongly depends on the chemical nature of the surface. The hydrophobicity of the surface is a major factor determining the surface influence on the free diffusion of the probe molecules near the surface.

• Korean Journal of Optics and Photonics
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• v.23 no.3
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• pp.113-118
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• 2012

The concentration and hydrodynamic radius of nano-sized fluorescence particles diffusing in solution were compared by using fluorescence correlation spectroscopy (FCS), which can measure the variation of the correlation function of a fluorescence signal by size and number of particles. The used nano-sized fluorescence particles are Alex Fluor 647, quantum dots, and fluorescence beads, and three kinds of sample solutions with different concentrations were prepared by dilution to 1/10 and 1/100 with distilled water for each kind of particles. The effective focal volumes were calculated by using the known diffusion coefficient of Alexa Fluor 647 particles, and the diffusion time, number of particles in focal volume, and variation of concentration according to the dilution could be measured by the FCS system. Through this study, we determined that the concentrations of arbitrarily diluted sample solutions can be measured by a home-built FCS setup in the range of 0.1 nM ~ 10 nM and that the diffusion coefficient of the quantum dot was $27{\pm}1{\mu}m^2/s$.

• 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.

• Journal of the Optical Society of Korea
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• v.12 no.3
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• pp.170-173
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• 2008

In this study, we simulated a statistical model of FCS(fluorescence correlation spectroscopy) based on a Poisson process to understand and explain observations of the experiment performed on molecules of ultra-low concentration by the home-built laser-scanning confocal microscope. The statistical model confirmed that the relative mean square amplitude of fluctuations is shown to be inversely proportional to the average number of molecules, even in the ultra-low concentration, if some conditions are satisfied. Signal-to-noise ratio and the variability of dwelling time under the confocal volume were found to be effective conditions for the experiment.