Fluorescence correlation spectroscopy single-molecule detection

The main luminescence parameters traditionally measured are the frequency of maximal intensity Vmax, intensity I, the quantum yield < >, the hfetime of the exited state T, polarization, parameters of Raman spectroscopy, and excited-state energy migration. The usefulness of the fluorescence methods has been greatly enhanced with the development of new experimental techniques such as nano-, pico-, and femtosecond time-resolved spectroscopy, single-molecule detection, confocal microscopy, and two-photon correlation spectroscopy. 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

Single-molecule fluorescence detection was subsequently demonstrated at room temperature, first by detecting the burst of light as a molecule passes through the focus of a laser beam [67,68], but each molecule could be detected only once in this way. Correlation analysis of many such bursts provides a window into a variety of dynamical effects ranging from diffusion to intersystem crossing to rotational correlation [69], and this area termed fluorescence correlation spectroscopy (FCS, ([70-72]) has been reviewed in [73]. 

R. Rigler, J. Widengren, Ultrasensitive detection of single molecules by Fluorescence Correlation Spectroscopy, in Bioscience, ed. by B. Klinge, C. Owman (Lund University Press, Lund, 1990), pp. 180-183... 

The nanosized detection area Ar or volume created by STED also extends the power of fluorescence correlation spectroscopy (FCS) and the detection of molecular diffusion [74,95]. For example, STED microscopy has probed the diffusion and interaction of single lipid molecules on the nanoscale in the membrane of a living cell (Fig. 19.6). The up to 70 times smaller detection areas created by STED (as compared to confocal microscopy) revealed marked differences between the diffusion of sphingo- and phospholipids [74]. While phospholipids exhibited a comparatively free diffusion, sphingolipids showed a transient ( 10 ms) cholesterol-mediated trapping taking place in a < 20-nm diameter area, which disappeared after cholesterol depletion. Hence, in an unperturbed cell putative cholesterol-mediated lipid membrane rafts should be similarly short-lived and smaller. 

Furthermore, novel methods for single molecule detection will contribute to improved drug discovery. For instance, fluorescence correlation spectroscopy (FCS) is characterized by the ability to measure single molecules in sub-micro liter sample volumes with a typical range of sensitivity between 10 6 and... 

The recently developed fluorescence correlation spectroscopy permits studies of molecular associafion in one femtoliter of solufion using a confocal or two-photon microscope. Two lasers are used to excite two fluorophores of different colors, each one on a different type of molecule. Fluorescence of single molecules can be defected, and molecular associations can be detected by changes in the distribution of fhe flucfua-tions in fluorescence intensity caused by Brownian rnohon. A different type of advance is development of compufer programs that analyze chromosomes stained with a mixture of dyes with overlapping spectra and display the result as if each chromosome were painted with a specific color. 

Fluorescence Correlation Spectroscopy and Single Molecule Detection... 

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