Immobilized single fluorescent molecules measurements

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. 

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.

Figure 6. Examples of the single molecule analyses enabled throu confocal fluorescence microscopy, (a) Fluorescence image of single molecules immobilized on a glass cover slip. TTie shs e and size of the fluorescence spots directly reflects die size and intensity distribution of the confocsd laser ot that excites the molecules. The image is a spatial map of the location of each molecules. After an images has been acquired, each molecule can then be addressed individually to measure (b) changes in spedrsd emission, (c) fluorescence lifetime or correlated emission of photon pairs (photon antibunching, see text), or (d) intensity transients.

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