Immobilized single fluorescent molecules

In this section we outline the two main types of single molecule measurement that we have chosen to discuss in detail in this text measurements on diffusing fluorescent single molecules and measurements on immobilized single fluorescent molecules. We introduce the basic concepts of these experiments, which we then expand upon in both a phenomenological and rigorous mathematical way in subsequent chapters. 

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. 

Figure 11.2 The image in the upper left panel shows a snapshot of several individual protein molecules immobilized in a gel. Each protein undergoes conformational fluctuations that can be monitored by a fluorescent probe. The fluorescent signal from a single protein molecule, as a function of time, is recorded in the time trace shown in the lower left panel. On the right, the experimental situation and the fluorescent time trace are idealized as a two-state conformational transition process as given in Equation (11.5), with A representing the darker state and B representing the brighter state. Image and data in left panel obtained from Lu et al. [133], Reprinted with permission from AAAS.

Fig. 27.3. Parallel orientation and diffusion of single TDl molecules in a highly ordered domain, (a) Sequence of fluorescence images showing linear diffusion of single TDI molecules in a chloroform atmosphere extracted from a time series. Scale bar 2 um. (b) Trajectory extracted from the molecule marked with the white circle in (a), (c) Calculated angular time trajectory of the same molecule, (d) Sketch of TDI molecules immobilized in the mesoporous film in air. The stars indicate active silanol groups, (e) TDI molecules in the mesoporous film in the presence of chloroform. The solvent provides a lubricant for the molecular movement...

Figure 8.18 Spatial patterns of fluorescence of single porphycene molecules A and B immobilized in poly(methyl methacrylate) at 293 K.

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.

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... 

Figure 3.6 (a) Schematic illustration of 2a reversible transition between the metal-free bright state (left) and the complexed dim state (right), (b) Fluorescence image of single 2a molecules immobilized on a... 

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