Sample preparation for Scanning electron microscopy (SEM)
SEM is primarily useful for giving a three‐dimensional image of the surface of the
specimen and is for viewing large objects.
As with TEM, specimens are imaged with a beam of electrons, but instead of the
electrons being transmitted through the specimen, the beam is "scanned" across,
creating an image of the surface of the sample, with exceptional depth of field. This
image is achieved via the detection of "secondary" electrons that are released from the
specimen as a result of it being scanned by very high energy "primary" electrons (ie.
those emitted from the electron "gun" in the SEM). As most biological specimens are
made up of non‐dense material the amount of secondary electrons produced is too low
to be of much use in creating an image and therefore they are usually coated with a very
fine layer of a metal which readily produces secondary electrons. The large depth of field
achievable can produce an image of great visual depth with a three‐dimensional
appearance.
The operating environment of a standard scanning electron microscope dictates that
specialist preparation techniques are used. Typically, a biological specimen is
chemically fixed, dehydrated through an acetone or ethanol series and then dried at the
critical point ‐ a method used to minimize specimen distortion due to drying tensions.
For dry samples, this process is not necessary. SEM can also be used to investigate
smooth surfaces of industrial samples.
The samples are mounted on a stub of metal with adhesive, coated with 40 ‐ 60 nm of
metal such as Gold/Palladium and then observed in the microscope.
Voltage Contrast
The interaction of a charged particle beam with structures of different electric conductivity in a microelectronic
circuit locally changes the electric potential at its surface. In a scanning electron microscope (SEM), this effect
leads to a distinct voltage contrast superimposed on the image. It is very useful for failure analysis and device
debugging in microelectronic research and development.
Introduction
Bulk insulating specimens, or insulated layers on a conductive
substrate with a thickness larger than the electron range,
charge up when irradiated by the electron beam in the SEM.
If the combined drain of backscattered and secondary
electrons exceeds the supply of primary beam electrons,
a positive net charge results that generates a positive electric
surface potential. The trajectories of primary, secondary
and backscattered electrons are influenced by this potential.
Part of the secondary electrons will be prevented from
reaching the detector. Thus, insulated structures will appear
darker in the secondary electron image than grounded
structures that do not charge (Fig. 1). This phenomenon
is called passive voltage contrast. Electrical opens, for instance
in contact chains, can be easily found with this method.
In integrated circuits, electric surface potentials can also
be actively controlled by applying voltages to selected
interconnects. The connected structures will then be visible
with different contrast in the secondary electron image.
Similar to passive voltage contrast, positive regions appear
dark, while negative regions appear bright (Fig. 2). This
active voltage contrast is used to detect dielectric leakage
or metallisation shorts. Moreover, active elements in the
device under test can be dynamically driven, enabling
waveform and timing measurements.
Active voltage contrast on microelectronic devices requires
precise probing using micromanipulators. Interconnects can
be contacted using a single microelectrode tip to apply a voltage
against the grounded substrate, and closed electrical circuits
can be constructed by contacting with two or more tips
simultaneously. A tip positioning accuracy in the nano-metre
range, low drift, low backlash, and insusceptibility to vibrations
of the micro-probing setup are indispensable for successful
use of this method on modern nanoscale interconnects.