Principles of Fluorescence Spectroscopy

764 SINGLE-MOLECULE DETECTION
Figure 23.11. Single-molecule images of oxazine 720 in poly(methylmethacrylate)
obtained using NSOM. Image size 4.5 x 4.5 µm.
Revised from [13].
spatial resolution is not limited to optical resolution and is
determined by the diameter of the fiber. In this image the
spots are below 100 nm in diameter.
SMD imaging is also measured using laser scanning
confocal microscopy (LSCM). LSCM is accomplished
using optics similar to Figure 23.6. Instead of observing a
single spot in the image, the laser excitation is raster
scanned so that the focused light is scanned across the sample.
29–31 The light collected by the objective is focused on
the pinhole to pass light from the desired volume element
and reject light from outside the focal volume. A singlepoint
detector, usually an SPAD, is used to measure the
light returning from each point in the image.
Single-molecule imaging has been accomplished using
standard epifluorescence and a high-quality CCD detector.
This is possible if the sample is thin, where there is little or
no signal from molecules outside of the focal plane. Figure
23.12 shows an epifluorescence image of a tetramethylrhodamine-labeled
lipid (TMR-POPE) in a monolayer of
POPC prepared as a Langmuir-Blodgett film. 36 Single molecules
were observable because the sample was only the
thickness of a phospholipid monolayer. This image illustrates
the need for extremely low fluorophore concentrations
for SMD. The mole fraction of TMR-POPE has to be
below about 10 –6 for single molecules to be observed
because two fluorophores closer than the optical resolution
of the microscope appear as a single spot.
23.4. INSTRUMENTATION FOR SMD
Prior to showing additional experimental results it is valuable
to examine a typical instrument used for these meas-
Figure 23.12. Epifluorescence images of TMR-POPE in a monolayer
of POPC. The mole fractions of labeled lipid are (a) 6.5 x 10 –3 , (b) 6.5
x 10 –6 , (c) 6.5 x 10 –8 , and (d) 6.5 x 10 –9 mole/mole. In (a) the intensity
was divided by 50. Exposure time was 5 ms. Reprinted with permission
from [36]. Copyright © 1995, American Chemical Society.
urements. Figure 23.13 shows the schematic for a confocal
SMD instrument. 37 Laser light is brought to the sample by
reflection off the dichroic filter. If needed, the polarization
of the incident light is adjusted with polarizers and/or wave
plates. Emission is selected by the same dichroic filter and
then passes to the detectors. In order to find and/or image
the fluorophores the stage is scanned in the xy direction,
Figure 23.13. Schematic for stage-scanning confocal SMD. Revised
and reprinted with permission from [37]. Copyright © 1998,
American Chemical Society.