Chemistry Reference
In-Depth Information
3.2.3
Darkfield Imaging with One Nanometer Accuracy (DIONA)
Dark eld microscopy is similar to the optical arrangement shown in Figure 3.1B
using a high numerical aperture condenser to illuminate the specimen with
high-angle glancing rays, but at lower angles relative to the optical axis so that the
incident light propagates obliquely into the sample compartment. The viewing
objective has a lower NA o than the condenser, so the oblique illumination is not
collected unless the light is scattered by the sample. Several groups have placed gold
nano-particles on their macromolecules which scatter light effectively, producing
bright spots on the dark background, similar to a fluorescent emitter, but not limited
by excited state lifetime, photobleaching or blinking [35, 36]. The number of collected
photons is limited only by intensity of the illumination and the scattering ef ciency of
the particle. With 40-nm gold aggregates,
s m ΒΌ
6 nm noise was obtained at 300-
m
s
sampling intervals. With 200-nm plastic beads a time resolution of 20
s was
achieved [37]. An application of this method to the events during myosin stepping
is described in conjunction with Figure 3.12.
m
3.2.4
Single-molecule High Resolution Imaging with Photobleaching (SHRImP)
Photobleaching can be made into an advantage for measuring the distance between
two fluorophores. When a sample molecule contains several identical uorescent
probes that are located too close together to resolve directly, they produce a PSF
similar to a single
fluorophore, but more intense (Figures Figure 3.3B
F and
-
Figure 3.5C). The two
fluorophores bleach sequentially (Figure 3.5B) and the point
spread function from the longer-lasting probe (panel D) can be subtracted from the
original two- uorophore distribution (panel C) to generate the PSF from the shorter-
lived probe (E). Fitting the two single- uorophore distributions to Gaussians locates
them with the usual FIONA precision, and the difference in positions gives the
original distance between the two probes. This SHRImP method complements
another more commonly used technique, to be described later, Fluorescence Reso-
nance Energy Transfer (FRET) for quantifying single molecule distances. Whereas
FRET between two spectrally matched fluorophores is ideal in the distance range
2.5 - 7.5 nm, SHRImP allows distances to be estimated at any separation, approxi-
mately 10 nmand above [38]. Application of thismethod tomyosin VI is given later in
this chapter. As described, the technique is limited to static measurements because
photobleaching is irreversible. This limitation might be avoided by applying
SHRImP to probes that blink.
3.2.5
Single Molecule Fluorescence Resonance Energy Transfer (smFRET)
When two fluorescent probes, such as Cy3 and Cy5, with overlapping emission and
absorption spectra are near to each other, the energy of optical excitation of the shorter
 
Search WWH ::




Custom Search