Single-molecule fluorescence spectroscop

Schuler, B, lipman, EA, Steinhach, PJ, Kunke, M, and and Eaton, WA, Polyproline and the spectroscopic ruler revisited with single-molecule fluorescence. Proceedings of the National Academy of Sciences of the United States ofAmericalOl (2005) 2754-2759. 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

The vast majority of single-molecule optical experiments employ one-photon excited spontaneous fluorescence as the spectroscopic observable because of its relative simplicity and inlierently high sensitivity. Many molecules fluoresce with quantum yields near unity, and spontaneous fluorescence lifetimes for chromophores with large oscillator strengths are a few nanoseconds, implying that with a sufficiently intense excitation source a single... 

Fluorescence resonance energy transfer (FRET) is a spectroscopic means of obtaining distance information over a range up to 80 A in solution. It is based on the dipolar coupling between the electronic transition moments of a donor and acceptor fluorophore attached at known positions on the RNA species of interest. It can be applied in ensembles of molecules, either by steady-state fluorescence or by lifetime measurements, but it is also very appropriate for single-molecule studies. In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer. 

The complete optical setup of induced fluorescence involves an excitation part and an emission part. The intersection point of the two parts is the detection window in the microdevice. The excitation part starts from the light source and ends at the microdevice, while the emission part originates from the microdevice and stops at the detector. In free-space fluorescence detection, the common excitation sources include the laser, light-emitting diode (LED), and mercury or xenon arc lamp, and each light source has its distinctive spectroscopic property and practical benefits. Laser-induced fluorescence (LIE) is a highly sensitive optical detection method and is able to perform even single molecule detection. LIE has been introduced into the... 

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