Diffusing fluorescent single molecules measurements

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. 

This work shows nicely that single molecule measurements on freely diffusing FRET labelled molecules can be used to provide information similar to that which an ensemble experiment would provide, but in addition allows subpopulations in a complex system to be resolved. Improvement in fluorescence probes to reduce photobleaching and conformational flexibility through the use of short, rigid linkers will be necessary to get the best results from this approach. 

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... 

In general, the correlation technique as well as the quantum jump technique are powerful tools to unravel complicated molecular photophysical dynamics for a single absorber. This statement is exemplified by the investigation of the chromophore terrylene, for which no kinetical parameters of the triplet state were known from ensemble measurements. The ISC rates presented here were determined solely by experiments on single molecules. Actually, it would be quite difficult to measure absolute rates of photophysical ISC parameters by other techniques when the triplet quant yield is smaller than 10 . Recently, fluorescence correlation spectroscopy was also proposed as an appropriate method for the determination of triplet parameters of fluorophores in solution [75]. Additionally, it is a helpful tool to investigate spectral diffusion of single absorbers as discussed in Sections 1.4 and 1.5. 

In the second type of experiment that measures single molecule spectral dynamics one performs repeated fluorescence excitation scans of the same molecule. In each scan the line shape is described as above, but now there is the possibility that the center frequency of the line will change from scan to scan because of slow fluctuations. Thus one can measure the center frequency as a function of time, producing what has been called a spectral diffusion trajectory. This trajectory can, in principle, be characterized completely by the spectral diffusion kernel of Eqs. (16) and (19), but of course it must be understood that only the slow Kj < 1 /t) TLSs contribute. In fact, the experimental trajectories are really too short to be analyzed with this spectral diffusion kernel. Instead, it is useful [11, 12] to consider three simpler characterizations of the spectral diffusion trajectories the frequency-frequency correlation function in Eq. (14), the distribution of frequencies from Eq. (15), and the distribution of spectral jumps from Eq. (21). For this application of the theoretical results, in all three of these formulas j should be replaced by s, the labels for the slow TLSs. 

---