Biomedical Engineering Reference
In-Depth Information
Transmission electron microscopes (TEMs) image the density of electrons. The
intrinsic absorption of every sample imposes to work on thin samples. Either it is
naturally the case for instance with molecules or membrane patches that are then
stuck on a thin carbon film, or thick samples have to be microtomed into thin slices
before their observation.
In a transmission microscope, areas richer in electrons appear darker in the
image. As the biological sample samples are composed of light elements (carbon,
oxygen, hydrogen) heavy elements have to be added to increase the contrast. It is
common to use chemical staining or gold nanoparticles functionalized with anti-
bodies that target particular proteins. They then appear as dark spots in the TEM
images specifically located on the molecules of interest.
If TEM works in the transmission mode, the scanning electron microscope
(SEM) works in the reflection mode. Again the parallel with the optical path of an
optical reflection microscope is tempting, the difference being that, in the present
case, a SEM does not form the image of the reflected electron beam but analyzes the
electrons scattered by the surface where the incident beam has been focused. Again
the optics are magnetic lenses.
These electron beam techniques have a very high resolution up to the point
were TEM-based techniques can resolve some protein structures. They however
need a sometimes tedious preparation of samples.
Atomic Force Microscopy
The atomic force microscopy (AFM) works with a completely different principle
[32]. Here, the surface to be analyzed is scanned under a fine stylus mounted on a
flexible leaf-spring (a cantilever) in the same way as a stylus probes the surface of LP
records in old-fashioned phonographs. When one is interested in microparticles or
macromolecules, the first step is to strongly adsorb them on this surface. The deflec-
tion of the cantilever is then a direct measurement of the topography of the surface.
To get some orders of magnitude, the radius of curvature at the apex of the tip is of
the order of a few tens of nanometers, the cantilever spring constant is of the order
of a few tens of mN/m. In most of the commercial instruments, not to say all of
them, the detection of the position of the cantilever is performed optically by shining
a laser beam on the back of the cantilever and measuring the reflected beam with
a quadrant photodiode. The relative displacements of the sample versus the tip are
performed by piezoelectric actuators in the three directions of space (Figure 8.16).
In practice, the mode just described where the vertical position of the sample is
fixed and the force of the tip acting on it varies, is seldom used for two main rea-
sons. First, by using the microscope this way, the force is higher on the ridges or the
bumps of the surface and lower in the valleys. As with any observation technique,
applying a force is already a potentially perturbative process (the extreme case be-
ing scratching the surface), but having different forces on the surface may make
the images very difficult to interpret. The second difficulty is more instrumental.
Getting true vertical distances from the measurement of the deflection of the canti-
lever would necessitate an accurate calibration of the detector for each experiment,
which is practically unreliable.
There is however a way to circumvent these difficulties, which follows a very
general instrumentation strategy: the force (given by the deflection of the cantilever)
