Single molecule fluorescence techniques molecules

This book is aimed at experimental scientists with a physical chemistry or biochemistry background who wish to enter this new and exciting field of research and to apply single molecule fluorescence techniques to studies of macromolecular structure and function. The book is designed to present an introduction to the topic, from the practical implementation of single molecule fluorescence experiments, through methods of data analysis to a description of a range of current and future... 

Abundant examples of diffusion spFRET can be found in the literature. Indeed it is in this area that single molecule fluorescence techniques have arguably proved the most useful. In Chapter 5 we review three papers in detail but here we present a very brief review of a broader range of spFRET experiments. 

As can be seen from Table 4.2, nucleic acids, proteins or heterogeneous complexes of one or both of these, have been the most commonly investigated biomolecules using single molecule fluorescence techniques. As no intrinsic naturally occurring fluorophores with suitable photophysical properties for single molecule experiments are found in either nucleic acids or polypetides (see Section 4.2.1), it is necessary to introduce such moieties into these biomolecules [5,24,25]. This can be done in three ways ... 

Optical properties of dendrimers bearing eight chromophores have been examined by single molecule spectroscopy techniques. It is especially exciting that variations in the spectra are recorded if one of these dendrimers is observed for a period of time under continuous irradiation.1481 The fluorescence intensity of the dendrimer nanoparticle also jumps between discrete emissive levels. All these findings suggest the existence of strong electronic interactions between several perylene imide chromophores within one dendrimer and provide new... 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

SECM instruments (77,78) will undoubtedly increase the scope and power of SECM. Further improvements in the power and scope of SECM has resulted from its coupling scanning probe or optical imaging techniques, such as AFM (57,79) or single-molecule fluorescence spectroscopy (80). The combined SECM-AFM technique offers simultaneous topographic and electrochemical imaging in connection to a probe containing a force sensor and an electrode component, respectively. 

One such approach has recently been developed and shown to enable high-resolution NSOM fluorescence and force measurements on viable cultured human arterial smooth muscle (HASM) cells under buffered conditions [28,29], This approach takes advantage of the nanofabrication capabilities of focused ion beam (FIB) milling to sculpt a light delivery structure into the end of a conventional AFM probe. The FIB technique, which utilizes a focused beam of gallium ions to mill samples with nanometer resolution, was first used by van Hulst and co-workers to modify conventional NSOM probes [30]. They demonstrated an improvement in single molecule fluorescence measurements using... 

Within the last years, single-molecule fluorescence imaging became possible at room temperature and ambient conditions, first with the use of scanning near-field optical microscopy and ultra-high spatial optical resolution (about 100 nm), and later with the use of diffraction limited microscopy techniques (resolution about 300 nm) such as the epi-fluorescence microscopy, internal reflection microscopy, and scanning confocal microscopy. 

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