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DONE BY STAFF OF EM UNIT
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INTRODUCTION
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Conventional SEMs require specimens that are
compatible with a high vacuum in order to operate
properly. The main reason for this is that
electrons need to be in a high vacuum in order to
travel in a straight line down the column without
scattering. Specimens that outgas in the high
vacuum of the chamber, or specimens that have
some degree of water in them (however small),
will degrade the vacuum to such an extent that
the SEM will not be able to operate. It is
especially important that the source of
electrons, the electron gun, is held under high
vacuum. Specimens that have some degree of water
content have to be processed in some way in order
to be able to image them in a conventional SEM.
Processing can be in the form of dehydration in
alcohols and a critical point dryer (CPD) or
simply to freeze dry the specimen. Freeze drying
requires the specimen to be frozen first and then
held under a vacuum until all the water is
sublimed. Both techniques are time consuming and
especially with CPD can lead to severe shrinkage
artefacts. Also, neither technique is very good
for viewing delicate samples, such as fungal
hyphae. Lastly, water in the sample can be frozen
and imaged with a cryo stage in the SEM. This
process does not result in artefacts but is time
consuming as the preparation chamber and SEM
chamber have to be cooled to liquid nitrogen
temperatures. A new kind of SEM was required to
be able to image specimens in a suitable
environment in the chamber whilst maintaining a
high vacuum in the electron emission area at the
top of the column. The main drive for this was
actually the wool industry in Australia that
required imaging of wool in its native condition
without preparation. Basically a column had to
be designed that would keep the electron beam in
a high vacuum environment as far down the column
as possible and only allow it to pass into the
sample environment close to the specimen,
therefore minimising beam scattering. This was
achieved by adding Pressure Limiting Apertures
(PLA) in the column and a differential pumping
system that could pump away any gas moving
through the apertures from the sample environment
and the high vacuum in the column. It was a
company called ElectroScan that first combined
the differential pumping system and PLA
technology to produce the first Environmental
Scanning Electron Microscope, or ESEM as it is
commonly known. The ESEM dual PLA pumping system
was able to create and maintain specimen chamber
pressure levels significantly higher than normal
SEM vacuum conditions, up to 20 TORR. The
introduction of the gaseous chamber environment
also gave rise to the need for detectors that
could operate in a gaseous environment.
Conventional SE detectors use a very high bias
voltage to attract the electrons and this cannot
be used in a water vapour environment.
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ESEM PUMPING TECHNOLOGY
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The main principal behind the ESEM technology
lies in separating zones of pumping so that
different levels of vacuum are produced and
controlled.
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This gas is pumped away by the rotary pump.
Separating this region from the high vacuum
region is another aperture and the high vacuum
region is pumped by a turbo or oil diffusion
pump.
The specimen chamber area can have pressures of
up to 50 TORR.
A small aperture separates this region from the
intermediate region and gas flows from the high
pressure to the low vacuum region.
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In high vacuum mode all of these regions are
pumped by the high vacuum pump and no apertures
are required. In low vacuum mode only the
upper aperture needs to be in place, but this
results in lower pressures being attainable. If
higher pressures were applied then too much gas
would flow through this aperture and the gun
emission area would suffer from poor vacuum.
True ESEM mode requires both upper and lower
apertures to be in place in order to attain the
higher pressures. It is the pressure limiting
apertures (PLA), which form the basis of the ESEM
technology, coupled with the special gaseous SE
detectors. In fact, the lower PLAs are built into
the detectors while the upper PLA is built into a
small, exchangeable device called an insert.
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ESEM DETECTORS
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Special detectors had to be designed in order to
operate in a gaseous environment. As there are
three pressure modes in the ESEM, three distinct
types of detector are needed to image secondary
electrons in these modes.
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EVERHART THORNLEY DETECTOR In high vacuum the
Everhart Thornley detector (ETD) operates by
creating a very high bias (300 volts) on its
Faraday cage. This pushes the low energy
secondary electrons towards a phosphor screen,
biased at 10kV, where they collide and produce
light. A photomultiplier tube amplifies the
signal which is then processed to form the SEM
image. In a water vapour environment which
occurs in low vacuum or ESEM mode, the 10kV
applied to the phosphor screen will cause the
detector to arc and malfunction. When switching
to either of these alternative modes the ETD will
be switched off.
HIGH VAC
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GSE DETECTOR When the primary electron beam
interacts with the specimen surface it produces
secondary electrons. These secondary electrons
will then interact with the water molecules in
the specimen chamber and knock off electrons
creating gaseous secondary electrons (GSE). At
the same time that GSEs are produced the water
molecules are being turned into positive ions.
These then migrate to the specimen surface where
they neutralize any charge build up on the
specimen surface. The GSEs continue to interact
with other water molecules and continuously knock
off electrons in what is known as gas
amplification.
ESEM
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The figure shows a typical gaseous secondary
electron detector which is now used in the latest
Quanta series ESEM systems. The detector is
simply plugged in at the back of the chamber and
the detector ring plugs over the insert (which
goes into the pole piece). The detector type is
automatically detected by the system so that no
confusion can occur on the pressures which can be
used. Careful observation of the detector will
reveal the PLA in the centre of the detector
ring.
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LARGE FIELD DETECTOR This other type of gaseous
detector is used in the low vacuum mode to image
specimens that are compatible with high vacuum
but are non-conductive. This detector does not
need the lower PLA and so only works in lower
pressure modes than the GSED. It is also detected
by the SEM automatically when inserted so that
the pressure will not exceed the allowable
limits. The LFD works in a similar way to the
GSE but needs a longer distance between the
sample and the detector in order to amplify
enough signal. As the detector works in a low
vacuum environment the primary beam is scattered
a lot less than in a higher pressure. This has
important implications for the user as the
quality of the image will vary significantly
according to the beam conditions, kV, working
distance etc.
LOW VAC
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IMAGING CONDITIONS
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Several conditions must be taken into account
when performing imaging in low vacuum or ESEM.
They all are inter-related and changing one
condition may also require changing another in
order to maintain a good image.
Temperature Accelerating voltage Pressure
Spot size Beam gas path length Scan speed
Click here to see examples
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TEMPERATURE
This condition refers specifically to the ESEM
mode as this is where the Peltier stage is used.
This is a specially designed stage for the ESEM
which can allow cooling of the specimen from
below 0ºC up to around 40ºC.
The Peltier allows for very specific
experimentation to be performed in the chamber,
such as wetting/drying experiments or
stabilisation of substances that are not stable
at room temperature. Another useful benefit of
the Peltier stage is that cooling the sample will
allow an increase in water content. Water is very
electron dense and a sample with high water
content will give a higher secondary electron
yield with better contrast than a sample with low
water content.
As the temperature and pressure around the sample
are lowered the water content will increase (or
surface water will increase if it is
non-permeable), resulting in a better quality
image (compare images 1 and 2)
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Image 1 25ºC, low contrast Image 2
5ºC, high contrast
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This figure shows the relationship between
temperature and pressure. As the temperature
decreases at any given pressure then the
proportion of liquid phase water increases in
relation to the gaseous phase. If the temperature
is decreased enough in a given pressure range
then the true ESEM condition is reached, whereby
liquid water will appear on the sample in the
chamber.
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PRESSURE
This figure also shows the relationship between
the pressure in the chamber and the water phase
present. Again, selecting a constant temperature,
for example 4ºC, and then lowering the pressure
in the chamber will result in the ESEM condition
being reached at about 5 TORR. As pressure is
more easily and quickly changed and controlled in
the ESEM it is usually this parameter which is
used while the temperature remains constant.
Pressure in the chamber also has a direct effect
on the imaging quality as it affects the number
of collision events the primary electron beam
will have with water molecules (see table) and
the strength of gas amplification.
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This table shows a variety of pressures that can
be used in the ESEM, from low vacuum through to
true ESEM mode of operation.
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In the low vacuum mode there is only one PLA
normally used (though two can be used) and it can
clearly be seen that increasing the pressure in
the chamber has a dramatic effect on the
proportion of the primary beam that reaches the
specimen in a focussed spot. The rest of the beam
is scattered away from the focussed spot and will
contribute to noise in the image. However, even
if only a small proportion of the beam remains in
the focussed spot then an image will be
attainable without loss of resolution. A long
acquisition time (ie slow scan) will be needed in
such a case to improve the signal to noise ratio
in the recorded image. When working in ESEM it
can be seen that the addition of the second PLA
(in this case an EDX cone at a distance of 2mm
from the sample) will have a significant effect
on the usable portion of the beam. Whereas 1
TORR with 1 PLA will result in 48 beam scatter,
this level of beam scatter only occurs at 10 TORR
if a second PLA is used in the ESEM mode. This
has advantages for imaging samples that require
higher pressures in order to maintain their
stability (eg wet, oily samples). Back to table
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If both beam scatter and gas amplification are
considered then there will be an optimum
condition whereby the scatter of the primary beam
and amplification of secondary beam result in
the best image. Ideally, beam scatter should be
at a minimum and gas amplification should be at
a maximum.
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Increasing the pressure will cause an increase in
signal up to a point. Once the maximum signal
level has been found, increasing the pressure
will result in a degradation of the signal due to
excessive beam scatter and an increase in the
noise content in the image. Other factors will
also normally play a part in choosing the correct
pressure, such as charging of the specimen and
stabilising the water content of the specimen.
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BEAM GAS PATH LENGTH
Beam Gas Path Length (BGPL) literally refers to
the distance that the primary electron beam has
to travel in a gaseous environment. In the low
vacuum mode there is only one PLA usually
inserted and this results in a limitation in the
lowest kV and/or the highest pressure that can be
used. Example
The final lens aperture (PLA1) is about 20mm
from the sample and so the BGPL is 20mm.
There is much scattering due to the long BGPL.
This limits the pressure to 1 TORR in a tungsten
SEM where 48 of the beam will still be usable.
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If the PLA2 is added then the maximum pressure
that can be used is increased as the BGPL will be
much less, thus much less beam scattering. With
the use of the conventional detector the BGPL
optimum will be at about 10mm. This allows for
a good proportion of the unscattered beam to
reach the sample while the gas amplification is
also good. However, this can be improved
further by the addition of a cone.
The BGPL is decreased to only 2mm and thus
minimise the beam scattering. The cone can be
used in either ESEM or in low vacuum mode. It is
useful in low vacuum mode as it will allow the
use of very low kV, low vacuum imaging.
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ACCELERATING VOLTAGE
Accelerating voltage (kV) will greatly effect the
quality of the image. Generally higher kV will
be required if high resolution is needed but this
results in increased beam penetration and/or
charge build up on the specimen. Even in low
vacuum an insulating specimen may still charge up
if too high a kV used. Switching to a lower kV
is the best solution but this then presents its
own problem a low kV beam will be scattered
more in the gas than a higher kV at any given
pressure. Again, the cone can help greatly in
this case as it will allow the BGPL to be much
reduced so more beam reaches the specimen. In
ESEM mode, where higher pressures are required to
obtain an image, the minimum kV that can be used
is about 10kV. In low vacuum mode this can be
down to 1kV with the cone. As a guideline When
in ESEM mode the optimum imaging conditions will
be with the cone in place, greater than 10kV and
a pressure of 2-3 TORR. When in low vacuum mode
the optimum imaging conditions will be with the
cone in place, greater than 2kV and a pressure of
0.7-1.0 TORR.
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This table shows how pressure, BGPL and kV are
all inter-related. The optimum image will be
attained with a BGPL of 2mm (with the cone) and a
pressure of 2 TORR. Raising the pressure to 5
TORR reduces the usable beam due to increased
scattering. With the cone in place and with a
pressure of 2 TORR the lowest kV that can be used
is about 2kV, at 5 TORR this is about 8kV. By
removing the lower PLA, as would be the case in
low vacuum, the lowest usable kV is about 10kV at
1 TORR.
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SPOT SIZE
Whereas accelerating voltage controls the speed
at which electrons travel down the column, spot
size controls the number of electrons that are
travelling. A small spot size is achieved by
having very few electrons in the beam in the
upper column. Few electrons (ie low beam current)
means that there are fewer electron-electron
interactions and the beam broadens very little as
it travel down the column, resulting in a small
spot. If more electrons are in the beam (ie the
beam current is higher) then there are more
electron-electron interactions and the beam gets
broader as it travels down the column, resulting
in a large spot. Smaller spots are necessary for
imaging at high resolution but as the number of
electrons is lower the overall signal level in
the image is lower. More noise will be visible
and the image will appear grainy. Selecting a
larger spot will help to a point but if too large
a spot is selected then the features of interest
on the image may no longer be visible too large
a spot size can also result in charging as the
current load will be too high.
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SCAN SPEED
The GSE detectors used in Low Vacuum mode and
ESEM mode need to have a good electron signal in
order to function properly. This is also the case
with the Solid State Backscatter Detector which
can be used in either high vacuum or low vacuum.
If a fast scan is used then the quality of the
image will suffer due to the noise level,
especially if a small spot is being used. A
slow scan means the beam dwells on any one pixel
for longer and the signal to noise ratio on the
pixel is improved. The overall effect is of a
cleaner image with a slow scan as opposed to a
fast scan. TV rate imaging is not possible as
the GSE detector simply cannot process the signal
that quickly.
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EXAMPLES
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How does the pressure and spot size affect the
imaging?
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What happens if a high pressure is used?
So if the pressure is reduced?
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How does the accelerating voltage affect the
image?
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How about using a lower kV?
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Does changing the spot size help in low kV
imaging?
What can be concluded from the examples?
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These are examples of images obtained at low kV
with the help of the CONE
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Using the cone at low kV and in low vacuum, these
cells show good surface details.
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Low kV in the ESEM mode and with the long cone
shows very good surface detail on these cells.
Pay close attention to the cell attachments.
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SUMMARY
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The use of the ESEM requires a thorough
understanding of what happens to the beam under
the many different conditions. Changing
parameters such as kV, pressure and scan speed
all have a direct effect on the quality of the
image and also what level of details can be
seen on the sample.
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• A general summary of operating conditions is
  given below
• Decreasing temperature will enhance contrast in
  most samples.
• Increasing pressure will stop charging but too
  much pressure will cause noise due
• to excessive beam scatter.
• A short beam gas path length is required to
  prevent excessive beam scatter and
• thus reduce noise levels in the image.
• So long as some beam remains in a focussed spot
  then resolution is not impaired
• even though noise levels may be higher.
• A large spot may produce a nice looking image
  but a non conductive sample may
• charge even in ESEM mode.
• A large spot will also reduce the potential to
  get a high resolution image. High
• magnification needs a small spot while low
  magnification can benefit from the low
• noise levels of a larger spot.