Counting and Behavior of an Individual Fluorescent Molecule without Hydrodynamic Flow, Immobilization, or Photon Count Statistics

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Abstract:

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Foldes-Papp (2002) Pteridines, 13, 73-82; Foldes-Papp et al. (2004a) J. Immunol. Meth., in press; Földes-Papp et al. (2004b) J. Immunol. Meth., in press) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame singlefluorescent- molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

Keywords: brownian motion; generalized continuous markov process; one and the same single; spatio-temporal stochastic behavior

Document Type: Review Article

DOI: http://dx.doi.org/10.2174/1389201043376986

Affiliations: Clinical Immunology and Jean Dausset Laboratory, Graz University M.S. and Hospital, Auenbruggerplatz 8, A-8036 Graz, LKH, Austria.

Publication date: April 1, 2004

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  • Current Pharmaceutical Biotechnology aims to cover all the latest and outstanding developments in Pharmaceutical Biotechnology. Each issue of the journal contains a series of timely in-depth reviews written by leaders in the field covering a range of current topics in both pre-clinical and clinical areas of Pharmaceutical Biotechnology. Current Pharmaceutical Biotechnology is an essential journal for academic, clinical, government and pharmaceutical scientists who wish to be kept informed and up-to-date with the latest and most important developments.
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