Principles of Fluorescence Spectroscopy

778 SINGLE-MOLECULE DETECTION
Figure 23.35. Typical configuration for single-molecule polarization
measurements.
reasonable when one recalls it takes considerable time to
scan the sample stage, so the molecule can rotate before the
stage returns to that molecule for a second illumination.
Another approach to single-molecule orientation imaging
is to use polarized excitation. As shown in Figure 23.33
the emission will show the same polarization independent
of the polarization of the incident light. Polarized excitation
was used with NSOM to image GFP in poly(acrylamide)
(Figure 23.37). Excitation was accomplished with an aluminum-coated
fiber optic with a 70-nm aperture using 488nm
laser light. 66 The relative intensities in the two polarized
channels were used to calculate the orientation of the tran-
sition dipoles on the slide. Since the emission remained
polarized during the scan, the GFP molecules were immobilized
by the poly(acrylamide) gel. The images show that
the individual GFP molecules each have different photostabilities.
Molecules 2 and 3 are stable and did not blink.
Molecules 4 and 5 displayed blinking. Molecule 1 appears
to have been photobleached during the scan.
Single-molecule orientations can also be determined
using NSOM and the shape of the single-molecule
image. 67–69 Figure 23.38 shows a focused-ion-beam (FIB)
image of the NSOM probe. The probe is coated with aluminum,
except for the small dark ring in the center (a). The
sample was DiIC 12 in PMMA. Image b shows the NSOM
images when the excitation is circularly polarized. The
images are not simple circles because of the sharp electricfield
gradients that exist near the tip of the metal-coated
fiber. Because of these field gradients the interactions of the
fluorophore with the NSOM tip depend on its location and
the orientation of the transition moments.
The orientation of the fluorophore can be determined
from the shapes, but the theory is rather complex. The lower
two images were obtained using linearly polarized light, as
shown by the arrows. All the images are color coded
according to the detected polarization. One of the circular
images in panel b becomes a double-labeled image with linearly
polarized excitation in panels c and d. These polarized
NSOM images provide remarkable detail on fluorophore
orientation.
23.8.2. Imaging of Dipole Radiation Patterns
Advanced Topic
The previous results on single-molecule polarization imaging
were obtained using point-by-point measurements and a
Figure 23.36. Polarized emission images of R6G molecules in poly(methylmethacrylate). The PMMA had a glass temperature of 8°C. The two orthogonal
polarization images (left and center) were used to construct the summed image (right). The white bar is 1 µm. Reprinted with permission from
[64].