Biology Reference
In-Depth Information
multiprotein system that has been studied for many years, providing a body of
knowledge from which we can now apply single-molecule methods to address
the physical principles that underlie the mechanism. It has only recently
become possible to image multiple proteins at the single-molecule level albeit
currently there are many limitations, such as protein labeling, excitation of
multiple dyes, and cross talk between multiple-emission color channels. How-
ever, with the introduction of Qdots that are all excited by a single wavelength,
the possibilities for multiplexing are rapidly becoming reinforced. This still
leaves key problems such as multiple specific protein labeling, and although
imaging multiple colors is challenging, the use of image splitting and recombin-
ing technologies has enabled multiple differentially filtered images to be
projected onto a single camera chip, permitting real-time multicolor imaging.
Furthermore, with the very recent commercialization of complementary metal-
oxide semiconductor (CMOS) cameras, the available field of view has increased
enormously without the cost of readout speed. Although issues of sensitivity
and quantum efficiency still loom before CMOS, the rapid advances in
EMCCD tell us that a new generation of highly sensitive rapid-imaging cam-
eras is around the corner. Much larger image chips enable more colors to be
imaged at the same time; three and four colors are currently possible with even
more being conceivable in the near future. These new cameras combined with
multicolor protein tagging and single-molecule tracking may allow real-time
imaging of all the steps of DNA repair processes in living cells in the future.
B. Overcoming the Brownian Motion Barrier
What happens as a diffusing molecule moves? We return to the problem
that it is not trivial to distinguish hopping from sliding. Evidence for both
scenarios exists but the definitive test is to directly observe motion along DNA
(see
Table I
). Currently, the time resolution for such motions is limited by
photon capture, that is, an image is formed by the photons collected by the
detector; more photons give a more precise estimation of the position of the
protein. However, more photons take longer to collect, in which time a diffuser
can move, reducing the accuracy of its position determination. This paradox
can be addressed by collecting photons more quickly; one way to do this is to
use dark-field microscopy. A gold nanoparticle attached to the protein of
interest will scatter light in proportion to the rate (intensity) of photons striking
it. If the incident light enters from an oblique angle, then it will not be directly
detected, thus creating a ''dark'' background. This approach has been success-
fully used to measure the motion of the molecular motor myosin V
104
with
submillisecond time resolution. By observing slowly diffusing DNA-protein
complexes moving with high spatial and temporal resolution, it will be possible
to resolve whether proteins slide or hop. Additionally, gold nanorods offer the
