• 제목/요약/키워드: Fluorescence correlation spectroscopy (FCS)

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Characterization of the Surface Contribution to Fluorescence Correlation Spectroscopy Measurements

  • Chowdhury, Salina A.;Lim, Man-Ho
    • Bulletin of the Korean Chemical Society
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    • 제32권2호
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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.

형광입자들의 크기와 농도에 따른 형광 상관 분광함수 측정 (Measurement of Fluorescence Correlation Function by Using Size and Concentration of Fluorescence Particles)

  • 한예슬;이재란;김석원
    • 한국광학회지
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    • 제23권3호
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    • pp.113-118
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    • 2012
  • 형광입자의 크기와 수에 따라 형광 신호의 상관함수 변화를 측정하는 형광상관분광법을 이용하여 용액 내에서 확산 운동하는 나노크기 형광 입자들의 농도와 유체역학적 반지름을 비교하였다. 시료에 사용된 나노크기 형광 입자들은 Alexa Fluor 647, 양자점, 형광 bead이고, 증류수에서 1/10, 1/100로 입자들이 들어있는 용액을 희석하여 각 입자들에 대해 3가지의 다른 농도의 시료를 준비하였다. Alex Fluor 647의 알려져 있는 확산시간을 이용하여 형광상관분광장치의 유효초점 부피를 구하고, 각 입자들의 확산계수, 크기, 희석에 따른 농도 변화를 측정할 수 있었다. 본 연구를 통해, 자체 제작된 형광상관분광장치로 임의적으로 희석된 시료들의 농도를 약 0.1 nM ~ 10 nM의 범위에서 측정할 수 있었고, 양자점의 확산계수를 $27{\pm}1{\mu}m^2/s$로 결정할 수 있었다.

Specific Binding of Nile Red to Apomyoglobin

  • Chowdhury, Salina A.;Lim, Man-Ho
    • 대한화학회지
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    • 제55권5호
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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.

Statistical Analysis of Fluorescence Correlation Spectroscopy of Ultra Low Concentration Molecules with a Confocal Microscope

  • Lee, Soon-Hyouk;Lim, Gyu-Chang;Kim, Soo-Yong;Kim, Eun-Kyung;Kim, Hak-Sung;Kim, Sok-Won
    • Journal of the Optical Society of Korea
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    • 제12권3호
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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.