Single molecule fluorescence experiments

Aitken, C. E., Marshall, R. A., and Pughsi, J. D. (2008). An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys.J. 94, 1826-1835. 

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... 

A number of common physical phenomena can affect and influence the autocorrelation function of a diffusion single molecule fluorescence experiment (Figure 2.1) and they are summarized in Figure 2.11. The principle component which generally dominates the autocorrelation function is diffusion (Figure 2.11(a)) [38]. The Stokes-Einstein relation describes the translational diffusion coefficient of a particle in a viscous medium,... 

Figure 2.11 Schematic showing of some of the processes that lead to fluctuations in a diffusion single molecule fluorescence experiment. White circles represent active fluorescent molecules (a) diffusion of a single labelled molecule in the inhomogeneous excitation volume, (b) triplet crossing causing intermittent fluorescence, (c) reversible binding with a second molecule that is not fluorescent but quenches the fluorescence of the labelled molecule, (d) conformational changes that induce changes in the amount of emitted fluorescence, and (e) photobleachlng.

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 3.1 Schematic overview of the elements of a single molecule fluorescence experiment.The italicized...

Since fluorescent dyes usually have quite broad emission spectra, narrow band pass filters that reject the majority of the unwanted background signals can also reduce the number of fluorescence photons reaching the detector. Minimizing the sample volume and the use of appropriate filters are the two most important approaches to improving the signal-to-noise ratio in single molecule fluorescence experiments. 

Figure 3.15 Illustration of the three filter types used in single molecule fluorescence experiments.

SINGLE MOLECULE FLUORESCENCE INSTRUMENTATION 127 Table 3.2 Summaty of light sources suitable for single molecule fluorescence experiments... 

Table 3.3 summarizes a range of microscope objectives suitable for single molecule fluorescence experiments along with some references giving examples of the single molecule applications in which they have been used. 

Table 3.3 Summary of some of the microscope objectives suitable for single molecule fluorescence experiments and examples of their most suitable (although not exclusive) application...

The choice of detector is another critical stage in the development of a single molecule fluorescence experiment. In general the detector should flalfil the following... 

In this section we review a number of important concepts that affect the choice of dyes for single molecule fluorescence applications. We introduce some concepts such as quantum yield, quenching, photobleaching, and blinking, many of which have already been highlighted in the context of single molecule fluorescence experiments and analysis in Chapters 2 and 3. We also refer the reader to a number of sources that provide further information on some or all of these topics [ 1-4]. 

The single molecule fluorescence experiments were carried out using a Confocorconfocalmicroscope (CarlZeiss, Germany).Nanocarriers (at 0.18 nM) labelled with the anti-estradiol antibody are loaded with fluorescently labelled... 

Chung, H.S., McHale, K., Louis, J.M., Eaton, W.A. Single-molecule fluorescence experiments determine protein folding transition path times. Science 335,981-984 (2012)... 

Much of the interest in SERS to date has focused on analytical applications. The technique has been used in a sensitive biosensor of glucose [72] and to assay lysophosphatidic acid, a biomarker for ovarian cancer [73]. With the advent of single-molecule SERS, biophysical studies comparable to the single-molecule fluorescence experiments described in Sect. 5.9 should be interesting. 

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