Chemistry Reference
In-Depth Information
frequently, so a physical sorting into different solutions is impossible (unlike cell
sorting). However, it does allow a brief time of
1ms to study the subpopulation
using other methods. In this way, we have used single-molecule sorting to facilitate
the measurement of
fluorescence lifetime curves of individual subpopulations, and
to analyze them in order to determine distance distributions that
uctuate on
nanosecond time scales using time-resolved FRET [44].
9.3.5.7 Trajectory Analysis of Single Molecules
The most striking data from single-molecule experiments show real-time conforma-
tional changes and transitions as a function of time. For transitions or changes on a
relatively slow time scale (
10ms or longer), there are a suf
cient number of
photons to produce a strong signal on time scales over which the system does not
change. At each of these time points, the state of the system may be determined
and followed as a function of time. All transitions or changes are observable
and determined. This simple analysis of time traces is commonly used for immo-
bilized molecules, and has produced striking results. Hidden Markov modeling [73]
and Bayesian models [74
-
76] may be used to obtain more precise results. For
marginal cases, where the signals are not so clear, these more advanced methods
are necessary to obtain proper results. Even in cases with strong signal to noise, these
methods are able to obtain more precise results with better de
ned error estimates.
9.3.6
Modeling and Simulations of Single-molecule Experiments
In order to properly understand the limitations of the experiments and determine
whether the analysis applied actually provides the information expected, modeling
and simulation of single-molecule experiments are necessary.
The modeling of nano-scale machines requires simultaneous modeling of deter-
ministic and random forces, as well as coupling to chemical reactions. Examples of
such modeling for molecular motors are reviewed in [4]. Polymer dynamics have
been modeled using molecular dynamics (MD) simulations [77], simple bead-spring
models [44] for analysis of single molecule data.
In addition to modeling the dynamics of interest, it is also necessary to model the
single-molecule
fluorescence experiments themselves. Modeling of single-molecule
fluorescence measurements requires coupling of such models to photophysical
simulations that include effects such as FRET, triplet states, saturation, etc. Modeling
of all of these features has been lacking, but some initial attempts may be seen
in [44, 72, 78].
9.4
Examples
In the remaining sections of this chapter we will discuss examples of single-molecule
experiments that relate to two classes of problems: intra-chain conformations i.e.
