Chemistry Reference
In-Depth Information
Immobilization of the system is of similar dif
culty to
fluorescent labeling,
requiring an attachment point on one of the molecules in the system. Successful
immobilization strategies include immobilization on coated glass surfaces, trapping
in immobilized vesicles, and immobilization in a gel matrix.
The primary bene
t of immobilization is the ability tomonitor onemolecule for an
extended period. This allows both the monitoring of equilibrium
fluctuations as well
as the response of a single molecule to triggered reactions or other non-equilibrium
events. In cases where solutions can be exchanged (surface immobilization), it is also
possible to wash away unused reagents during the preparation of the sample. The
drawback is the additional work of immobilizing the molecules, and the possibility
that the immobilization strategy will affect the function of the molecules.
In experiments using freely-diffusing molecules,
fluorescence is observed
brie
y(
1ms) from each molecule as it diffuses through a small observation
volume. Many molecules can be observed with this approach during a relatively
short experiment, and can be sorted into several subpopulations. No immobiliza-
tion strategy is needed, so the only changes to the molecules are the
uorophores,
thus minimizing potential problems. Sample preparation for each experiment is
much simpler and does not require any steps for immobilization. The main
drawback is the short observation time. Equilibrium measurements in solution
provide snap shots of the states of many molecules. By invoking the ergodic
theorem, stating that the behavior of an ensemble of molecules is equivalent to the
behavior of a single molecule over very long time periods, we can determine the
states accessible to each molecule.
Even in cases where immobilization is necessary, it is often desirable to perform
preliminary experiments on freely-diffusing molecules. This helps
fine tune experi-
mental conditions for single-molecule measurements, verify that
uorophores do
not affect protein function, and can often answer parts of the question or alternative
issues related to the primary question. Comparing the results of the equilibrium
distributions obtained from solution-based measurements and the smaller number
of long time trajectories obtained, immobilized measurements can be used to
demonstrate ergodicity in the system.
As discussed in the next section, small observation volumes are used in single-
molecule spectroscopy; these small volumes allow freely-diffusing molecules to
diffuse out of the observation volumes quickly (typically near 1ms). If longer
observation times are necessary to answer the experimenters question, it will be
necessary to immobilize the molecule.
9.3.3
What Excitation/Optical Isolation Format should be used?
In order to obtain single-molecule detection,
fluorescence from individual molecules
must be isolated from all other
fluorescence and scattering sources. Suf
ciently
ef
cient excitation and detection of a molecule is necessary to detect typically several
thousands of photons from a
fluorescent molecule per second. A small optical
detection volume must be obtained, minimizing background fromRaman scattering,
