Biology Reference
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medium as an evanescent wave that can excite fluorescent molecules. The light
intensity penetrating into the medium falls off exponentially with distance
from the coverslip. The exact depth of penetration depends on the angle of
incidence, the refractive indices of the two media and the wavelength of the
light. In practice, the propagation distance into the distal medium (the
distance over which the intensity falls by 1/e) can be less than 100 nm (for a
more detailed description see Toomre and Manstein, 2001). The emitted
fluorescent signal can be detected either from below through the same
objective used to launch the evanescent wave or from above using an
immersion objective.
The distinct advantage of TIRF is the very thin optical sectioning achieved
(5100 nm), which is significantly better than on confocal systems (4250 nm).
However, TIRF is not a replacement for confocal or other fluorescence
imaging techniques since it is not able to penetrate into a sample and 3D
imaging is not possible. In fact, TIRF is often combined with conventional
wide-field epi-fluorescence microscopy in order to relate surface effects to
internal cellular structures (Merrifield et al., 2000).
The very thin optical sectioning is critical to both applications of TIRF. For
viewing ventral cell surface processes, the exponential decay of illumination
intensity restricts fluorescent emission to the cell surface. For single molecule
fluorescence studies, the same limited illumination depth dramatically reduces
background fluorescence, increasing the signal-to-noise ratio and allowing
discrimination of single fluorescent molecules.
Two elegant examples of TIRF applications that show its versatility
involve imaging the invagination of clathrin-coated pits and measuring the
oligomerization of E-cadherin on the free cell surface. In the first example a
fusion protein, clathrin-dsRed (a red-coloured fluorescent protein), is imaged
at the ventral membrane using TIRF. The associated proteins, dynamin and
actin, are also imaged using either TIRF or conventional epi-fluorescence
enabling the study of the dynamics of the invagination process (Merrifield et
al., 2002 and Figure 7.2A and B). In the second example E-cadherin-GFP
fusion proteins were introduced into cell lines lacking E-cadherin. The
expressed fusion protein was detected in the ventral cell membrane by TIRF
and the intensity of fluorescent spots was measured. It was found that the
intensity was quantized, one quantum being the intensity due to a single
GFP molecule. The single molecule detection sensitivity was further
demonstrated by single step photobleaching from the single quantum
intensity level. The intensity of individual spots was then used to assess the
oligomerization state of the E-cadherin complexes showing that E-cadherin
oligomerizes before binding to its partner in cell-cell junctions (Iino et al.,
Finally, Justin Molloy's group (unpublished data) have used TIRF to
follow single molecules of GFP fused to the PH domain of myosin X. This
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