Chemistry Reference
In-Depth Information
showing that very near NA
c
1.33, the penetration depth of the evanescent wave
is several hundred nanometers, but in the usable range of incident directions,
NA
e
¼
1.4
-
1.49, d is approximately 100 nm.
The intensity of the evanescent wave at the interface, I
0
, depends on polarization of
the incident illumination. The plane containing the incident and re
ected beams is
termed the scattering plane, the x
-
z plane in Figures 3.1 and 3.2. s-Polarization is
de
ned as excitation linearly polarized (with its oscillating electric
field) perpendi-
cular to the scattering plane. p-Polarization is excitation linearly polarized parallel to
the scattering plane. The intensity of the evanescent waves at the interface for the two
polarizations are
¼
¼
s
E
2
4
n
1
cos
2
n
2
¼
s
E
2
4
n
1
NA
e
n
1
ðq
Þ
1
s
I
0
ð
3
:
2
Þ
n
1
n
2
¼
p
E
2
4
n
1
cos
2
2n
1
sin
2
n
2
Þ
n
1
ð
n
1
NA
e
Þð
2NA
e
n
2
Þ
ðq
Þð
ðq
Þ
1
1
n
1
n
2
¼
p
E
2
4
p
I
0
n
1
sin
2
n
2
NA
e
þ
n
1
NA
e
n
2
cos
2
ðq
Þþ
ðq
Þ
n
2
n
1
n
2
n
1
ð
1
1
3
:
3
Þ
where
s
E and
p
E are the electric
field magnitudes for the two polarizations. The
expressions for p-polarization are more complex than for s-polarization because
p-polarized excitation generates an elliptically polarized evanescent wave, explained
further in the Section 3.2.7. Plots of these functions (Figure 3.3B) show that the
p
I
0
is
approximately30%greater than
s
I
0
(when
s
E
p
E), theyaregreatestnearNA
c
, andthey
both decrease nearly linearly as NA
e
increases. In the range of NA
e
¼
1.4
-
1.49, the
evanescent wave intensities are approximately 1.5
-
2.5-fold greater than
s
E
2
and
p
E
2
.
For general imaging and localization of individual
uorophores, the polarization of
the evanescent wave should not select particular molecules over others. Therefore,
the incident beam is often circularly polarized. If the positions of lens L1 and mirror
M1 are reversed, and M1 and the objective back focal plane are both one focal length,
f
L1
, away from L1, then instead of moving L1, rotating M1 causes parallel translation
of the beam inside the microscope objective. This arrangement provides a means
for rapidly altering the incident angle which, in turn, alters the intensity and
penetration depth.
Figures 3.7, 3.9 and 3.14 show images of single
¼
fluorophores labeling various
molecular motor proteins. Each bright spot of
fluorescence in the images is
determined to be an individual
fluorophore or several
fluorophores on one molecule
based on their surface density as the quantity loaded on the surface is varied, the
distribution of
fluorescence intensities (single component, two components, etc.)
and sudden decrease of the
uorescence to a background level due to photobleaching.
The location, spatial orientation, interactions of these spots with other components of
the system and response to mechanical forces are parameters that can elucidate the
functional mechanisms of the molecules under study. The quality of these signals
depends critically on the brightness, steadiness andmaintenance of the
uorescence
emission.
Bright
fluorophores, ef
cient, high-aperture collection optics, high transparency
emission
filters, the quantum ef
ciency of generating charge carriers per photon
