Chemistry Reference
In-Depth Information
field) and the absorption dipole moment. When a polarizer is introduced before a
narrow aperture detector, the likelihood of registering the emitted photon is given by
cos
2
e
is the angle between the
fluorophore emission dipole and the
detector polarization. In xanthene-derivative dyes, such as rhodamine, and cy-dyes,
such as Cy3, the absorption and emission dipoles are aligned with the long axis of the
chromophore.
Several groups have used excitation light circularly polarized in the plane of the
microscope slide (the x
q
e
, where
q
fluorescence emission polarized
along the x and y axes [15, 49]. With this optical arrangement, the ratio of intensities
between the two detectors, corrected for any differences in their sensitivity, gives a
signal, independent of total intensity, but sensitive to the angle (
y plane) and separated the
-
f
) of the emission
of 45
or
-
45
,
the two recorded intensities are equal; angular discrimination away from45
or
-
45
depends on differences between the two intensities. For a given average angle,
rotational mobility (wobble) of the probe or of the macromolecule causes the two
recorded signals to becomemore equal, so the signals aremixtures of themean angle
and extent of mobility. This optical arrangement does not provide information
regarding the
fluorophore angle (
dipole projected onto the x
-
y plane. At
fluorophore angles giving
f
q
p
) relative to the optical axis, because both
the exciting and detection polarizations are in the x
-
y plane. Nevertheless,
interesting and useful information about motions within molecular motors were
obtained [15, 49].
3.2.7
Polarized Total Internal Reflection Fluorescence Microscopy (polTIRF)
In polTIRF, the orientation of the
fluorophore is detected by illuminating the sample
with linearly polarized light at various known angles and resolving the polarization of
the
fluorescence emission. In our implementation of this scheme, Pockels cells are
used to rapidlymodulate the path and polarization of a laser beam in an objective-type
or prism-type TIRFmicroscope (Figure 3.1). Four different combinations of scatter-
ing plane (either x
-
z,ory
-
z) and polarization (either s-orp-) are applied during
successive 5
-
10-ms intervals. In Figure 3.6, the x
-
z direction is indicated as Path 1
and y
-
z direction is labeled Path 2. For each of these input directions, the s-polarized
excitation beams produce x-ory-polarized evanescent waves. The p-polarized
excitation beams produce strong z-polarized evanescent waves with small, out-
of-phase components (5
-
10% intensity) along the x or y directions [11]. These four
combinations of optical path and polarization give evanescent waves polarized along
all three Cartesian coordinates (Figure 3.6). In a typical experiment on molecular
motors, cycles of the four combinations are completed every 20
-
40ms by alternating
the voltages on the Pockels cells.
A bright water-immersion objective, a polarizing prism, and two avalanche
photodiodes (APDs) collect the x-polarized and y-polarized
fluorescence emission.
The photon counts from the two APDs during each of the four intervals of input path
and polarization are binned into eight temporal traces,
s1
I
x
,
s1
I
y
,
p1
I
x
,
p1
I
y
,
s2
I
x
,
s2
I
y
,
p2
I
x
, and
p2
I
y
, that contain the angular information of the probe with the 20
-
40-ms
