Chemistry Reference
In-Depth Information
of photons originating from a dipole emitter is given by sin
2
e
is the
angle between the path of the photon and the emission dipole (Figure 3.7B). Photons
are most likely to travel perpendicular to the dipole and they are not emitted along the
dipole axis. An out of focus image is similar to sectioning the sin
2
w
e
, where
w
e
contour
slightly above or below the x
-
y plane, giving an asymmetric distribution of intensities,
containing lobes and fringes and an intensity gap in the direction of the dipole.
In DOPI, images of the
fluorophore are recorded approximately 0.5
w
m
m out of
focus and the resulting asymmetric intensity distributions are
ttedwith theoretically
predicted models (Figure 3.7C) [55]. The detected image of the single
uorophore
depends on
z from the focal
plane. DOPI enables relatively straightforward determination of the full hemisphere
of dipole orientations with useful angular and temporal resolution. Compared with
polTIRF, the instrumentation in DOPI is simpler to implement and can also
simultaneously determine the probes spatial position, a feature that has only
been partially implemented with polTIRF [73]. The temporal resolution of DOPI
(
q
and
f
as in polTIRF, and the amount of defocusing
d
0.5
-
1 s) is lower than that of polTIRF (40
-
80ms), but in many cases the biological
reaction can be slowed down tomatch the time resolution of themethod, for instance
by reducing the substrate concentration [56]. The angular resolutions of the
two techniques are comparable (
10
-
20
). PolTIRF, however, is sensitive to the
rate and amplitude of rotational wobbling motions of the probe and the labeled
macromolecule, whereas DOPI and similar techniques provide the average angle
over the recording gate time. DOPI has been applied to the stepping of myosin V, as
described later.
3.3
Molecular Motors
The single-molecule imaging techniques described above are readily applicable to the
understanding of the dynamics and energetics of the transport machines of the cell.
Some of the molecular motors that have been studied with single molecule imaging
are shown in Figure 3.8. Myosin V is a 450-kDa isoform of the myosin superfamily
having two heavy chains each with an N-terminal ATP- and actin-binding motor
domain, six peptide sequences, termed IQ motifs, each of which bind a calmodulin
subunit or a calmodulin-like light chain, and a tail that carries the dimerization and
cargo-binding domains.
Myosin V and some 20 other the myosin isoforms translate along actin to power
muscle contraction and cell shape changes, locomotion, cytokinesis, intracellular
vesicle transport and other motile, architectural and signaling functions. Muscle
myosin, designated myosin II, is similar to myosin V in having two globular
N-terminal motor domains, and a tail that dimerizes into an
a
-helical coiled coil.
In muscle, the tails of
300 myosin II molecules self-polymerize into bipolar
filaments that align sideways in the muscle to form the so-called A-band of the
sarcomere, the contractile organelle. Myosin II contains two IQmotifs that carry light
chain subunits structurally related to CaM.
