Biomedical Engineering Reference
In-Depth Information
Where
N
is the average number of fluorophores in the observation volume,
S
0
is a
geometrical
parameter, and
τ
0
is related to the diffusion coefficient
D
through the
relation:
τ
0
1/ D
Other relaxation modes can also be accessed using this technique, still at a very lo-
cal subcellular scale.
We have in the preceding part insisted that imaging of single particles was rela-
tively straightforward with sensitive enough detection equipment. By using this
idea, several techniques have emerged in the last decade that reconstitute a complete
image by the addition of many single object images. As the Rayleigh diffraction limit
is not relevant for single object imaging, the resolution that can be obtained can be
extremely good. Stimulated emission depletion (STED) microscopy was historically
the first of these so-called superresolution microscopies. Here, by using nonlinear
effects, the size of an activated fluorescent spot is decreased down to a few tens of
nanometers. Many of these spots are collected to form an image [29] improving the
resolution by an order of magnitude compared to the Rayleigh law.
Photoactivated localization microscopy (PALM) and stochastic optical recon-
struction microscopy (STORM) are two techniques that image dispersed nanoob-
jects. By choosing the right excitation only a few of them are randomly excited.
Their precise localization is then extracted from the image and the procedure is
repeated. The full image is then reconstructed from the superimposition of many
of these single molecule images (up to millions of them). The drawback here is the
dynamics. These operations take a long time, they are well suited to fixed cells on
which a resolution down to 20 nm can be obtained but not to dynamic processes
[30, 31].
Many developments are underway in this area. One of the next foreseen im-
provement will be to adapt these techniques to 3-D imaging while keeping the same
resolution; another one, as mentioned before, is to make them faster mainly by us-
ing other fluorophores, to observe dynamical phenomena.
8.3.1.3 Nonoptical Microscopies
Electron Microscopy
Electron microscopes use an electron beam to probe objects. The transmission elec-
tron microscope works on a similar principle as the optical microscope using elec-
trons and not photons. Because of the much smaller wavelength of the electrons,
the resolution obtained with these instruments is several orders of magnitude better
than with optical microscopes (down to a fraction of a nanometer). The optical
path is exactly the one used to describe optical microscopes: The source of elec-
trons is a heated filament, and the deflection of the electron beam corresponding to
the deflection of light by glass lenses is obtained by magnetic fields. Contrarily to
the optical microscope, the resolution is never the theoretical resolution imposed
by the Rayleigh formula (8.4) but is caused by aberrations inherent to the magnetic
lenses. Because of the interactions of the electrons with air, the whole setup is placed
under vacuum. This limits the applications of electrons microscopes: no live cell or
even hydrated sample can be imaged with such instruments.