Biomedical Engineering Reference
In-Depth Information
by modeling the field distribution, simulation of the absorption intensity with polarized
excitation, and comparison with data obtained at two different excitation polarizations (16).
NSOM has also been used to study dynamics, spectroscopy, and resonance energy transfer
of single molecules (11).
Near-field scanning optical microscopy suffers from several limitations. There is
unavoidable silica Raman scattering from optical fiber probes. A diffuse luminescence
emission is always generated from the aperture of the fiber probe. The preparation of the
probe is difficult to reproduce and sometimes the sample is perturbed by the presence of
the probe. It is interesting to note that single-molecule experiments have not yet been
reported at the highest theoretically possible resolution that should be achievable with
near-field method (33). This may be due to the weak interaction between a single molecule
and the radiation field, and therefore a greater power field may be required (while still
maintaining room temperature conditions).
2.3.3
Far-Field Confocal Microscopy
Laser scanning confocal fluorescence microscopy is particularly attractive due to its com-
bination of relatively large fluorescence (signal) capture and concurrent reduction of back-
ground signal (noise). In CM, pinhole plates are used to define and limit the depth of focus
as shown in Figure 2.1, rejecting “out-of-focus” radiation from areas beyond the focal
plane (37). Typically a laser source is reflected from a dichroic mirror (sometimes dichroic
beam splitter), into a large numerical aperture microscope objective that can focus excita-
tion radiation to a spot of approximately a 1 µm diameter on the sample. Fluorescence
from the sample is collected by the same microscope objective and passed by the dichroic
mirror through a pinhole located before a photon detector. The combination of high spa-
tial and depth resolution defines an excitation volume of 0.5-1.0 fL in the sample (25,26).
A cylindrically shaped excitation volume is typically about 0.5 µm in diameter and 2 µm
in height, which is determined by the diffraction limit for the wavelength of visible light
used, and the spherical aberration of the microscope objective.
Some of the first articles describing confocal fluorescence microscopy to detect single
molecules were published by Rigler and coworkers (30,38). They recorded the fluores-
cence from single rhodamine-6G molecules in water diffusing through a 0.24-fL volume.
The signal-to-noise ratio was determined to be about 1,000. With the development of
photon-counting systems, Nie et al. (39,40) applied confocal fluorescence microscopy to
study fluorescently labeled proteins and DNA fragments directly in real time.
Furthermore, with the improvement of laser excitation power, it has even possible to trap
and manipulate single macromolecules, such as DNA (11), either in microchannels or
using an approach termed “optical tweezers” (Figure 2.2). A number of research groups
have successfully applied these techniques to detect and identify single molecules (41-46).
As one example, the binding of green fluorescence protein (GFP)-labeled XPA protein to a
cyanine-labeled DNA substrate was assessed quantitatively by simultaneous detection of
both fluorophores using confocal fluorescence microscopy. Colocalization of cyanine and
GFP signals within one diffraction-limited spot indicated the formation of a single com-
plex of XPA with DNA (8). The use of microcapillary, microstructure, or monomodal
waveguide techniques have been provided for improved detection efficiencies, and have
reduced the need to operate using very dilute sample concentration conditions (11).
The advantages of far-field CM include reduction of background interference, unlimited laser
throughput (mW to W), three-dimensional sectioning capability, noninvasive detection, high
sensitivity, and experimental simplicity. However, because a confocal laser beam probes only
a single small excitation volume at a time, it is time-consuming to scan large sample areas of
