Diffusing fluorescent single molecules

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. 

Reilly P D and Skinner J L 1993 Spectral diffusion of single molecule fluorescence a probe of low-frequency localized excitations in disordered crystals Phys. Rev. Lett. 71 4257-60... 

Osborne M A, Balasubramanian S, Furey W S and Klenerman D 1998 Optically biased diffusion of single molecules studied by confocal fluorescence microscopy J. Chem. Phys. B 102 3160-7... 

Osborne, MA, Balasubramanian, S, Furey, WS, and Klenerman, D, Optically biased diffusion of single molecules studied by corfocal fluorescence microscopy. Journal of Physical Chemistry B 102 (1998) 3160-3167. 

Lyon and Nie [7] confined single molecules in a pulled silica capillary of submicrometer dimension (500 - 600 nm inner diameter) and detected single molecules with a con-focal fluorescence microscope. They found that the diffusion of molecules in the silica capillary was much slower than that in bulk solution, allowing 50 - 100 times longer observation period. Foquet et al. [10] reported a nanofluidic system made by microfabrication for fluorescent single molecule detection in 2004 and demonstrated that nanochannels could be used to isolate a single molecule for fluorescent detection. Tegenfeldt et al. [8] measured the... 

Ambrose W P and Moerner W E 1991 Fluorescence spectroscopy and spectral diffusion of single impurity molecules in a crystal Nature 349 225-7... 

Direct observation of molecular diffusion is the most powerful approach to evaluate the bilayer fluidity and molecular diffusivity. Recent advances in optics and CCD devices enable us to detect and track the diffusive motion of a single molecule with an optical microscope. Usually, a fluorescent dye, gold nanoparticle, or fluorescent microsphere is used to label the target molecule in order to visualize it in the microscope [31-33]. By tracking the diffusive motion of the labeled-molecule in an artificial lipid bilayer, random Brownian motion was clearly observed (Figure 13.3) [31]. As already mentioned, the artificial lipid bilayer can be treated as a two-dimensional fluid. Thus, an analysis for a two-dimensional random walk can be applied. Each trajectory observed on the microscope is then numerically analyzed by a simple relationship between the displacement, r, and time interval, T,... 

The sample is continuously irradiated and the fluctuations in the fluorescence intensity arise due to any event which makes the fluorophore unavailable to be excited to the emissive singlet excited state, such as diffusion of the fluorophore out of the detection volume, formation of a dark state, such as a triplet excited state, or photoreaction. The concentration of fluorophore in the detection volume has to be low (10 13—10 8M) so that the fluctuation in the intensity for one molecule is observable over any background emission. The high concentration limit is a consequence of the fact that the correlated photons from single molecules scale with the number of molecules in the detection volume, while the contribution from uncorrelated photons, arising from the emission from different molecules, scales with the square of the number of molecules. The lowest concentration is determined by the probability of finding a molecule in the detection volume.58... 

Luminescence is sensitive enough to observe single molecules.13 Figure 18-15 shows observed tracks of two molecules of the highly fluorescent Rhodamine 6G at 0.78-s intervals in a thin layer of silica gel. These direct observations confirm the random walk of diffusing molecules postulated by Albert Einstein in 1905. 

Aqueous phase (2.7 mm3) was placed in the thin lower compartment of the microcell and the Dil dodecane solution (63 mm3) was added on top of the aqueous layer. Fluorescence of the interfacial Dil was observed in the range of 571-575 nm. The influence of two kinds of surfactants, sodium dodecyl sulfate (SDS) and dimyristoyl phosphatidylcholine (DMPC), on the lateral diffusion dynamics of single molecules at the interface was investigated. DMPC was dissolved in chloroform, and the solution was mixed with pure diethyl ether at a ratio of 1 19 (chloroform diethyl ether) by volume. Pure water was placed in the lower container, and the DMPC solution was subsequently (5 mm3) spread carefully on the water. After evaporation of chloroform and diethyl ether, the Dil dodecane solution was added on the DMPC layer. Since Dil has a high... 

FIGURE 10.7. Total internal reflection fluorescence microscopy of the micro-two-phase system of dode-cane/water. (a) Continuous photons with an average of 11 molecules in the observation area, (b) photon burst with an average of 0.02 molecules in the observation area and (c) extension of the photon burst upon the addition of DMPC. A model for the photon burst upon the observation using single molecule diffusion (d). 

Fig. 2.5. Examples of single-molecule spectral diffusion for pentacene in p-terphenyl at 1.5 K. (A) A series of fluorescence excitation spectra each 2.5 s in duration spaced by 0.25 s showing discontinuous shifts in resonance frequency, with zero detuning = 592.546 nm. (B) Trend or trajectory of the resonance freqnency over a long time scale for the molecule in (a). For details, see [34]...

Single-molecule fluorescence detection was subsequently demonstrated at room temperature, first by detecting the burst of light as a molecule passes through the focus of a laser beam [67,68], but each molecule could be detected only once in this way. Correlation analysis of many such bursts provides a window into a variety of dynamical effects ranging from diffusion to intersystem crossing to rotational correlation [69], and this area termed fluorescence correlation spectroscopy (FCS, ([70-72]) has been reviewed in [73]. 

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