Simultaneous Characterization of Lateral Lipid and Prothrombin Diffusion Coefficients by Z-Scan Fluorescence Correlation Spectroscopy

Abstract In the present investigation, fluorescence con-elation spectroscopy (FCS) was used to measure the molecular motion of the pteridine derivative neopterin. However, technical limitations in the present optical setup precluded the identification of ,single neopterin molecules. FCS measurements with a fluorophore were also can-ied out for comparison. Exemplified by rhodamine green, we have introduced a concept that allows the detection, identification and analysis of assays in solution at the single-molecule level in tenns of bulk concentration. This concept is based on FCS and Poisson distribution analysis of assay sensitivity. The molecules had not to be quantified in a more concentrated fonn, or in flow and trapping experiments. The study demonstrated an ultrasensitive, reliable, rapid and direct tool for analytics and diagnostics in solution. We discuss a possible application of our new concept in activation control of cell-mediated immunity via neopterin determination.

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Monte Carlo simulation of fluorescence correlation spectroscopy data

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc2009526 ◽

2011 ◽

Vol 76 (3) ◽

pp. 207-222 ◽

Cited By ~ 3

Author(s):

Peter Košovan ◽

Filip Uhlík ◽

Jitka Kuldová ◽

Miroslav Štěpánek ◽

Zuzana Limpouchová ◽

...

Keyword(s):

Monte Carlo Simulation ◽

Monte Carlo ◽

Diffusion Coefficients ◽

Fluorescence Correlation Spectroscopy ◽

Rotational Diffusion ◽

Correlation Spectroscopy ◽

Analytical Models ◽

Spherical Particles ◽

Fluorescence Signal ◽

Fluorescence Correlation

We employed the Monte Carlo simulation methodology to emulate the diffusion of fluorescently labeled particles and understand the source of differences between values of diffusion coefficients (and consequently hydrodynamic radii) of fluorescently labeled nanoparticles measured by fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS). We used the simulation program developed in our laboratory and studied the diffusion of spherical particles of different sizes, which are labeled on their surface. In this study, we focused on two complicating effects: (i) multiple labeling and (ii) rotational diffusion which affect the fluorescence signal from large particles and hinder the analysis of autocorrelation functions according to simple analytical models. We have shown that the fluorescence fluctuations can be well fitted using the analytical model for small point-like particles, but the obtained parameters deviate in some cases significantly from the real ones. It means that the current data treatment yields apparent values of diffusion coefficients and other parameters only and the interpretation of experimental results for systems of particles with sizes comparable to the size of the active illuminated volume requires great care and precaution.

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