Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 767
Figure 23.17. Single-molecule images of DiIC 12 on glass obtained
using the stage-scanning configuration shown in Figure 23.13.
Revised and reprinted with permission from [43], Copyright © 1998,
American Institute of Physics.
portionality of number density to concentration and observation
of single-step photobleaching. The different intensities
for the various single molecules are thought to be due
to different orientations of the fluorophore relative to the
polarized incident light.
Close examination of the middle panel in Figure 23.17
shows that some of the single-molecule spots are half-circles
rather than circular. This result shows an important
aspect of SMD, all the fluorophores display blinking.
Examples of the blinking are shown in Figure 23.18 for
three different DiIC 12 molecules. In each case the intensity
fluctuates dramatically, and eventually the emission stops
when the molecule undergoes permanent photodestruction.
The middle panel is the most typical blinking profile: a relatively
constant intensity, followed by a rapid drop in intensity,
followed by a return of the intensity. This blinking phenomenon
explains the half-circles in Figure 23.17. Recall
that the stage is scanned in the x and y directions to create
the image. A single molecule is illuminated several times as
the sample is raster scanned through the laser beam. If the
fluorophore undergoes blinking or destruction during this
somewhat continuous illumination it is not seen in the subsequent
line scans, resulting in the partial circles. This illustrates
an important advantage of stage scanning with
SPADs. One can follow a single molecule in time to obtain
a more detailed view of its photophysical behavior.
An important feature of SMD is the ability to examine
the behavior of a single molecule rather than the average
Figure 23.18. Time-dependent intensities of DiIC 12 on glass. Revised
and reprinted with permission from [43], Copyright © 1998,
American Institute of Physics.
behavior of numerous molecules. This property is revealed
by the emission spectra for individual molecules of DiIC 12
(Figure 23.19). These spectra are not for the same molecules
used for Figure 29.18. Different emission spectra
were found for different molecules. Often the single-molecule
spectra were the same as the bulk phase spectra (lower
left and dashed). However, the spectra of single molecules
can also be dramatically different (right panels). These
spectral changes can be the result of a number of mechanisms,
such as different local environments on the glass.
The behavior of the molecules depends upon the composition
of the sample. The blinking and spectral changes are
usually different for fluorophores on glass or embedded in
a polymer. In the case of DiIC 12 in PMMA, different emission
spectra were observed for the same fluorophore at different
times, 16 a phenomenon called spectral diffusion. The
important point is that SMD can provide resolution of
underlying molecular heterogeneity, which can be due to
either the local environment of the fluorophore or the conformational
heterogeneity in macromolecules.
Single-molecule images of DiI-C 18 embedded in poly-
(methylmethacrylate) were also observed using NSOM. 44
The excitation was circularly polarized to provide equal
excitation of each fluorophore irrespective of the dipole orientation
around the z-axis. The emission was passed
through a polarizing beamsplitter to separate the polarized
components and sent to separate SPAD detectors. The spots
are color coded to show the dipole orientation, which was
determined by the relative intensities of each polarized
component (Figure 23.20). It is interesting to notice that
single molecules display polarized emission, even if the