Biomedical Engineering Reference
In-Depth Information
optics, for example the use of external reflection (16). TIRM is an effective wide-field alter-
native to epi-illumination to realize this design advantage.
2.3.5
Total internal Reflection Microscopy
Total internal reflection microscopy is a wide-field technique that can be used to observe the
interface between two media with different refractive indexes, such as glass and water (49).
The experiment is based on a near-field optical effect. An excitation laser beam enters a wave-
guide and is trapped in a medium of high refractive index, which is surrounded by a medium
of low refractive index if the beam is launched at an incident angle greater than the critical
angle of TIR as defined by Snell's Law (50). A standing (i.e., nonpropagating) evanescent field
appears at the interface of the two media and the intensity exponentially decays into the
medium of lower refractive index. The wavelength of the radiation in the evanescent field is
the same as in the waveguide, and the exponential decay of intensity limits the excitation
depth to a range of a few wavelengths, with most excitation energy being located close to the
surface of the waveguide (few hundred nanometers of visible radiation). The evanescent
wave allows for excitation of single molecules on (or near) the surface of the waveguide.
While the total energy in the evanescent field is often a very small fraction of that pumped
into the waveguide, the optical configuration substantially reduces background signals based
on scatter and emission from bulk solution. Finally, emission from single molecules is col-
lected by a microscopy objective and is typically imaged using an ICCD camera.
Hirschfeld is regarded as the pioneer of TIRM to detect single molecules. Early work
involved the detection of targets such as a single antibodies that were each labeled with
tens of fluorophores. With the advent of more sophisticated detection and sample manip-
ulation technologies, single fluorophores can be detected. Examples include detection of
labeled myosin and kinesin molecules (21,51,52), and visualization of cell signaling pro-
teins including peptide hormones, membrane receptors, small G proteins, cytoplasmic
kinases as well as small signaling compounds after labeling (36). A three-dimensional
image of a single Nile red dye molecule confined in nanometer-sized pores of polyacry-
lamide gels has been obtained by TIRM (53). Although TIRM has been used to study sin-
gle molecules that can freely move in solution, it is clear that more information can be
obtained if the molecules are immobilized (15).
A variety of geometries for TIR excitation of fluorescence have been used near a dielectric
interface in wide-field microscopy. Strategies include use of prisms, optical fibers, gaps
between optical slides, and use of multimodal and monomodal waveguides (54). The largest
signal-to-noise ratio can be obtained by using prism-TIR, while the largest total number of
detected photons can be obtained using prism-less through-objective TIR (54). No matter
which configuration is used, single molecule detection with TIR excitation seems more effec-
tive than that with wide-field epi-fluorescence because of lower optical background
contributed by out-of-focus excitation volumes and due to interfacial excitation (9,55-58).
2.4
Applications of Single Molecule Detection
Single molecule detection has the potential to explore nanoenvironments at a molecular
level. Single molecule allows exploration of heterogeneity and dynamic state changes with-
out synchronization in condensed phases (15). DNA sequencing, DNA fragment sizing,
protein conformations, and molecular dynamics have been addressed by single molecule
detection methods. Note that one important distinction to always consider when developing
