Immobilized single molecule experiments

Analysis and application of immobilized single molecule experiments... 

In general, neither the force nor the molecular extension can be controlled in the experiments so definitions in Eqs. (96), (98), and (99) result in approximations to the true mechanical work that satisfy Eqs. (40) and (41). The control parameter in single molecule experiments using optical tweezers is the distance between the center of the trap and the immobilized bead [88]. Both the position of the bead in the trap and the extension of the handles are fluctuating quantities. It has been observed [94—96] that in pulhng experiments the proper work that satisfies the FT includes some corrections to Eqs. (97) and (99) mainly due to the effect of the trapped bead. There are two considerations to take into account when analyzing experimental data. 

One of the big problems in single-molecule experiments is how to keep the molecule in place long enough to make observations on it, as well as how to grab hold of different parts of the molecule in order to manipulate it. In AFM and similar techniques the molecules are immobilized by being adsorbed on or attached to macroscopic surfaces. Another method relies on the properties of finely focused laser beams to act as optic il twcezersor optical trap-s to manipulate microscopic particles suspended in solution. 

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... 

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... 

Figure 3.9 Photostability of BODIPY dyes in a single-molecule experiment, (a) The images show single, immobilized BODIPY fluorophores at different times points which were recorded by a TIRF microscope upon...

To achieve single-molecule fluorescence detection, there are three common experimental practices. First, experiments are done at low concentrations (10 9-l() 12 mol I ) to spatially separate molecules, so each of them can be studied without interference from surrounding molecules. Second, fluorescence signal detection is confined to a small volume (<10-151) to minimize background noises for single-molecule sensitivity. Third, biomolecules are often immobilized, so a single molecule can be studied over time. 

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. 

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