Biomedical Engineering Reference
In-Depth Information
protein folding, DNA transcription, and singlet-to-triplet crossing rate determinations
(11,29). Applications of FCS in the area of biosensor development have included the deter-
mination of the mobility and function of “receptors,” such as association and dissociation
constants of intermolecular reactions. For example, when a short-labeled nucleic acid probe
associates with a larger unlabeled molecule, the diffusion rate of this labeled probe becomes
slower and the diffusion time is longer (29). According to the diffusion characteristics, the
binding fraction could be calculated from the correlation mathematics (29).
There are a number of other optical methods that can make use of stochastic and corre-
lation mathematics. Single molecules to be detected in flow cell when a dilute liquid sam-
ple flows through a confined volume that is excited by laser beam. Fluorescence is collected
at 90 or 180 degrees with respect to the direction of the laser beam (11), and the fluorescence
signal corresponds to single molecules passing across the excitation beam. This method can
be used in rapid DNA sequencing, optical sizing of DNA fragments, rare-event detection,
and ultrasensitive screening of combinatorial chemical libraries. This technique is operated
as a compromise that limits large sample volume throughput and high efficiency for single
molecule detection. An alternative to use of flowing streams or large volumes of static solu-
tion is to detect single molecules in microdroplets. One single molecule can be confined in
one picoliter-sized droplet. An electrodynamic trap is used to levitate the droplet so that the
molecule can be interrogated by a laser excitation for a longer period (11). The time required
for levitation and fluorescence measurement tends to be too long to allow this methodol-
ogy to be practical for single molecule counting and sorting.
2.3.2
Near-Field Scanning Optical Microscopy
Understanding of the organization and behavior of many basic molecular systems requires
an in situ analysis of conformational dynamics at the single-molecule level. NSOM provides
the ability to study single biomolecules without the ensemble averaging effects of traditional
far-field optical spectroscopy (Figure 2.1). NSOM overcomes the Rayleigh limit of optical
resolution and operates to provide spatial resolution in the range of 20-200 nm. The method
relies on excitation using a standing evanescent field from a small probe tip, and is sensitive
and well suited to study the dynamics studies of protein structure and conformational
change (31,32). When a localized source of radiation having an intensity profile that is much
smaller than its wavelength is placed within a fraction of one wavelength distance to the
interested surface, then the excitation area will be similar to the size of the localized source
(33). The source of radiation is usually a tapered optical fiber where the sides are coated with
metal to prevent light leakage (16). Finally, the optical signal is detected by using an objec-
tive lens in either the transmission or collection mode. As one example, an aluminum-coated
tapered optical fiber with a subwavelength aperture (5-10 nm) has been used as a wave-
guide for excitation using optical laser excitation, and can be raster-scanned at nanometer
distances across a sample (34). Shear-force feedback (11), as found in related methods such
as AFM, was used to maintain the tip at a constant distance from the sample, resulting in col-
lection of a spatially resolved dataset that provided topographic reconstruction with
nanometer resolution. Fluorescence that was emitted by individual molecules was collected
by an oil immersion lens of high numerical aperture, and signal was developed using a high-
sensitivity avalanche photodiode detector (34).
High-resolution NSOM has allowed the imaging of a fluorophore-labeled actin network
associated with intact fibroblast cells, and the results were deemed to be far superior to those
of the best far-field confocal images. Biomembrane imaging remains one of the frontiers of
near-field research (35). NSOM has been used to determine the dipole orientation of indi-
vidual fluorophores (16). The orientations of individual emitting dipoles were determined
