The confocal microscope
allows the investigator to visualize fluorescence from optically
thin sections by rejecting light from visual planes that are out
of focus, in essence increasing the signal-to-noise ratio. We
have used this technique to record fluorescence polarization from
a single fluorophore that was attached to a well-defined location
on the myosin molecule (Warshaw et al., 1998). To limit the number
of myosin molecules and thus fluorophores that are detected, we
have focused the light from an Argon laser to a diffraction-limited
spot on the motility surface. Thus the size of the spot on the
surface that is excited by the laser is approximately twice the
wavelength of light being approximately 1 µm in our experiments.
We used circularly polarized light to maximize the probability
that a single fluorophore will be excited. The fluorescence emission
was then split into its orthogonal components and passed through
a pinhole to reject out of focus light. Silicon avalanche photodiode
detectors that could count single photons were used to detect
the light. With this instrumentation, we determined the orientation
and motion of the neck region of myosin by estimating changes
in fluorescence polarization of a single fluorophore attached
to the myosin regulatory light chain.
