Chemistry Reference
In-Depth Information
Figure 3.15 Photo Activated Localization
Microscopy (PALM). A, a summed-molecule
(equivalent to standard) TIRF image of focal
adhesions for a cell expressing vinculin tagged
with the photoactivatable fluorescent protein,
dEos. B, magnified inset of adhesion region in A.
C, summed-molecule TIRF image near the
periphery of a cell expressing tdEos-tagged actin.
D, magnified PALM view of the actin distribution
within the box outline in C. Inset, further
magnified view. E and F, summed-molecule TIRF
and PALM images, respectively, of a cell
expressing dEos-tagged, retroviral protein Gag.
Considerable detail in the PALM image is not
resolved in the standard image. From Ref [179].
3.4
Conclusions
Studying individual molecules using novel types of optical microscopy is
helping to reveal the functional mechanisms of the molecular motors, myosin,
kinesin and dynein inways not thought feasible only a few years ago. Singlemolecule
biophysics avoids the inevitable loss of information in classical ensemble experi-
ments when a signal is averaged over the members of the sample population.
Fluctuations, reversals, rare events and other heterogeneities are the necessary
consequence of the stochastic nature of chemical and physical reactions. This noise
is interesting! Many of the signals that are the primary functional output of molecular
motors, such as piconewton forces and nanometer motions, are not accessible in a
collection of molecules suspended in a cuvette. Techniques have evolved for
localization of molecules at far better precision than the classical resolution of light
microscopy and for detecting structural dynamics such as rotational motions and
intramolecular distances. The experiments are both resolving questions and raising
further questions about these nano-machines: how they produce force andmotion,
how they transducemetabolic energy intowork and how they are regulated. Although
