Single molecules immobilized

Kim, H., Nienhaus, G., et al. (2002). Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules. Proc. Natl. Acad. Sci. USA 99(7), 4284-4289. 

Studies of immobilized single molecules can help to answer some of these questions. In these experiments the time available to monitor the molecule in the small observation volume is extended by immobilizing the molecule of interest, either tethered to a substrate (generally a glass slide. Figure 1.2(b)) orby immobilization in a gel or tethered liposome. In this way the observation time is only limited by the stability of the instrument used, the signal to noise ratio of the... 

Figure 1.2 Illustration of the concept of measuring the fluorescence from an immobilized single molecule, (a) A molecule, which can undergo a reversible transition between folded and unfolded conformations, is labelled with a dye at one terminus and a quencher at the other. In the folded (native) conformation the fluorescence from the dye is quenched. In the unfolded (denatured) conformation the fluorescence is enhanced. (b)The molecule is immobilized (tethered) onto a solid substrate and the fluorescence signal from a small volume near the surface monitored as a function of time. (c)The same molecule can be monitored for a considerable length of time and the stochastic transitions (the number of which depend on the height of the energy barrier for the transition) can be obsen/ed. Eventually (at around 9.5 s in this simulated example), photobleaching of the dye occurs to a non-fluorescent state, at which point no more information can be extracted from this molecule.

Practical considerations when studying immobilized single molecules... 

Analysis and application of immobilized single molecule experiments... 

In Chapter 6, we review a number of studies that illustrate the many ways in which single molecule fluorescence trajectories may be manipulated to give insight into many types of behaviour. Immobilized single molecule fluorescence experiments are now receiving considerable attention in the literature and new and exciting... 

Fluorescence spectroscopy of immobilized single molecules examples... 

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