ELECTRON MICROSCOPY

TECHNIQUES
Introduction
Electron microscopes are very powerful tools for visualizing biological samples.
They enable scientists to view cells, tissues and small organisms in very great
detail. However, these biological samples can’t be viewed on electron
microscopes whilst alive. Instead, the samples must undergo complex
preparation steps to help them withstand the environment inside the microscope.
The preparation process kills the tissue and can also cause changes in the
sample’s appearance. In this regard, every effort is made during preparation not
to alter the sample’s geography.

There are two reasons as to why living samples can’t survive the environment in
an electron microscope:
 The power of the electron beam that is directed at the sample.
 The vacuum inside the microscope.

The electron beam inside a transmission electron microscope (TEM) causes

problems for biological samples because of its high energy. It needs to have
enough energy to pass right through the sample and out the other side. The
temperature can get up to 150°C where the beam hits the sample. This
temperature is far too high for living cells to survive. Scanning electron
microscopes (SEMs) use a lower-energy electron beam, but it can still be
damaging to the sample.

The vacuum inside an electron microscope is important for its function. Without
a vacuum, electrons being aimed at the sample would be deflected (knocked off
course) when they hit air particles. But liquid water, which is abundant in
biological samples, evaporates immediately in a vacuum. If this happened, a
biological sample would vaporize.

And therefore, to be able to be visualized with an electron microscope,

biological samples need to be:
 Fixed (stabilized) so the electron beam doesn’t destroy them
 Dried thoroughly so the vacuum doesn’t affect them.
Sample Preparation for TEM
In this regard, samples to be observed under a transmission electron microscope
are prepared in this sequence:
i). Fixation
ii). Dehydration
iii). Infiltration
iv). Embedding
v). Sectioning and
vi). Staining

Fixation
Fixation of tissues is the most crucial step in the preparation of tissue for
observation in the transmission electron microscope. Fixation consists of two
steps:
 Cessation of normal life functions in the tissue (killing) and
 Stabilization of the structure of the tissue (preservation).

The goal of fixation is to preserve structure as faithfully as possible compared to

the living state. There are three very important parameters that one needs to put
in mind while carrying out fixation:
i). Keep the time between killing and fixation to a minimum.
ii). Keep the size of the tissue as small as possible without losing information
or destroying the tissue. If a large specimen must be fixed, keep one
dimension less than 1 mm, or else nick areas of the specimen that can be
discarded so that the fixative can penetrate. Effective penetration of
fixative is about 0.5 mm for osmium.
iii). Keep gross tissue deformation to a minimum by using sharp
implements and keep manipulation of the specimen to the minimum
necessary.

Every effort must be made to ensure that the tissue is kept moist in a
physiological medium until placed in a fixative. Dissecting in fixative can be
used if manipulation of the specimen is time consuming.
If the specimen floats, it must be submerged. In some cases, this may be done
by dipping the specimen in a wetting agent briefly before fixation or adding a
wetting agent to the fixative solution. If a wetting agent is added to the fixative,
then try to transfer the specimen to fresh fixative as soon as possible. Since
trapped air is often the cause of tissue floating in the fixative, air can sometimes
be removed by partial vacuum. However, this is sometimes damaging to the
specimen. The most gentle approach is simply to put a plug of Kimwipe® (Wipes
that easily wipe up dust and liquid) below the surface of the fixative, thereby trapping the
specimen beneath the surface of the fixative solution.

Once the specimen is fixed, use the same vial throughout preparation.
Simply decant (or pipet) the contents of the vial before each change. An
aspirator can be used to speed this process. Since tissue lost in the aspirator is
irretrievable, beginners would be advised not use the aspirator until they are
experienced with handling material.

Fixatives are best used fresh. Assume that all fixatives are at least mildly
photoactive and that all are sensitive to oxidation in the environment. For this
reason, fixatives are kept in the refrigerator or freezer and warmed to room
temperature just before use. Commercially prepared solutions are packed in
nitrogen gas. They include:

a) Glutaraldehyde (GA) which is normally clear and has a sickly sweet

smell. It can degrade into glutaric acid which is light yellowish in color.
Glutaraldehyde fixation can take place at room temperature or in the
refrigerator for 2 hours (for extrememely small material), 6 hours
(standard) or more if necessary. Some people hold material in GA for
weeks to years, and if it degrades the material, the degradation may not
be obvious. It fixes by cross-linking proteins.
b) Osmium tetroxide is a straw-colored crystal, which when mixed with
water results in a similarly colored solution.

It should only be used in a fume hood. It is extremely expensive, so each

sample should use only 1 - 2 ml of solution during fixation. It is highly
 labile at room temperature and all glassware used in its preparation
should be acid-washed prior to use to remove organic compounds that
will cause its breakdown. Osmium tetroxide in the presence of light, heat,
or organic materials will be converted to osmium dioxide, a black
compound which is ineffective at fixing materials. Used osmium and
black osmium solutions should be discarded in a special waste container
in the fume hood. Osmium vapors are extremely effective at fixing
mucous membranes and should be regarded as a hazard in the lab. All
osmium fixation should be conducted in the cold for up to 2 hours -- no
longer. Osmium tetroxide fixes lipids by cross-linking them.
c) Formaldehyde fixes proteins by cross-linking them.
d) Glutaraldehyde-formaldehyde mixtures
e) Acrolein
f) Tannic acid
Dehydration
Dehydration is the chemical removal of water from the specimen. Common
dehydrating fluids are ethanol and acetone. The potential problems of
dehydration are shrinkage of the specimen, plasmolysis, and removal of soluble
components from the specimen. Dehydration must be conducted relatively
rapidly in order to prevent excessive extraction of alcohol and acetone-soluble
compounds, but slow enough to prevent plasmolysis.

Extraction of specimen components is difficult to control. Low molecular

weight carbohydrates are particularly susceptible, since carbohydrates are
usually poorly cross-linked if at all following fixation. Proteins tend to be cross-
linked by glutaraldehyde during primary fixation and the lipids by osmium
tetroxide during secondary fixation. The carbohydrates are essentially unfixed.
Linked to the problem of extraction is that of shrinkage. Both problems are most
serious at low concentrations in the dehydration series. In general, rapid
dehydration is best for these reasons.

By 70% alcohol, the tissue no longer shrinks as much, but does begin to harden.
In fact, extended periods of dehydration in higher concentrations of alcohol may
make the tissue quite brittle. If a stopping point is needed, most histologists
choose 70% to 100% alcohol as a good place to stop for the evening.
If there is evidence of plasmolysis, perhaps additional dehydration steps (and/or
longer changes) may be required. Cell membranes sometimes retain some
osmotic activity after short periods of fixation. Longer periods of fixation in
glutaraldehyde can reduce osmotic sensitivity as well. (Membranes are
essentially insensitive to osmotic changes after 48 hours of fixation in
glutaraldehyde.) Poor fixation will aggravate problems with dehydration.
Dehydration at refrigerator temperatures slows the process down a bit and tends
to lend some rigidity to the tissue. It may also reduce plasmolysis slightly.
Plants are the most sensitive to poor dehydration, and therefore, refrigerated
dehydration is preferred for these tissues.

Infiltration
Infiltration is the replacement of the dehydrating fluid or transition solvent with
plastic resin. In this case, Epon 812 and Spurr's resin is used. Changes of 1/3
resin, 2/3 resin, and 100% resin are used in succession. The steps in infiltration
are fewer because less damage is done to the specimen during this stage. The
goal of infiltration is simply the complete penetration of resin into the specimen.
Solutions of resin and solvent are prepared immediately before use and added to
the specimen vial. Waste resin solutions are discarded in a special container in
the fume hood. The resins can be kept in the freezer between uses; however,
they must be allowed to warm thoroughly before opening the container since
water will condense on cold resin.
Use of slow rotation will aid penetration of the resin into the tissue block. The
quality of penetration can be judged only after embedding. In general, a slightly
longer period of infiltration is better than shorter period of infiltration.

Embedding
Embedding involves the final positioning of the tissue specimen in the liquid
plastic and its subsequent polymerization (hardening). To do this, the tissue is
passed through a 'transition solvent' such as propylene oxide (epoxypropane) or
acetone and then infiltrated with an epoxy resin such as Araldite, Epon, or
Durcupan. Tissues may also be embedded directly in water-miscible acrylic
resin. After the resin has been polymerized (hardened) the sample is thin
sectioned (ultrathin sections) and stained – it is then ready for viewing.
Three types of molds are commonly used in embedding: (1) small capsules, like
gelatin capsules or special plastic BEEM (Ballistic Electron Emission) capsules; (2)
flat embedding molds; (3) flat dishes.
Each has its own set of advantages and disadvantages. Embedding is the crucial
step in determining the orientation of sectioning. BEEM and gelatin capsules
are ideal when the specimen has no polarity, or when the specimen will orient
itself by gravity. For particulate (relating to or in the form of minute separate particles )
samples, special BEEM capsules with steep walls and a narrow tip are available.
Flat embedding molds are preferred when the specimen must be oriented in a
particular position in the liquid plastic. This allows accurate cross and
longitudinal sections to be prepared and helps in aligning the specimen. The flat
dish is useful when the specimen is large, when orienting the specimen is
impractical, or when massive amounts of material are scanned to select a
sample to section. The small plastic chip containing the desired specimen must
then be sawed out of the block and glued to a dowel or piece of cast plastic for
sectioning.

Sectioning
Sectioning for TEM infers to the cutting of thin sections (biological specimens
that are <100 nm thick). These thin sections allow electrons to pass and stop
only where stains delineate objects of interest. This is achieved using a
technique called ultramicrotomy.

Ultramicrotomy is a method for cutting specimens into extremely thin slices,

called ultra-thin sections, which can be studied and documented at different
magnifications in a transmission electron microscope (TEM). It is used mostly
for biological specimens, but sections of plastics and soft metals can also be
prepared. Sections must be very thin because the 50 to 125 kV electrons of the
standard electron microscope cannot pass through biological material much
thicker than 150nm. For best resolutions, sections should be from 30 to 60nm.
This is roughly the equivalent to splitting a 0.1mm-thick human hair into 2,000
slices along its diameter, or cutting a single red blood cell into 100 slices.
Ultramicrotomy process
Ultra thin sections of specimens are cut using a specialized instrument called an
"ultramicrotome".

The ultramicrotome is fitted with either a diamond knife, for most biological
ultra-thin sectioning, or a glass knife, often used for initial cuts. There are
numerous other pieces of equipment involved in the ultramicrotomy process.
Before selecting an area of the specimen block to be ultra-thin sectioned, the
technician examines semi-thin or "thick" sections range from 0.5 to 2µm. These
thick sections are also known as survey sections and are viewed under a light
microscope to determine whether the right area of the specimen is in a position
for thin sectioning. "Ultra-thin" sections from 50 to 100nm thick are able to be
viewed in the TEM.

Tissue sections obtained by ultramicrotomy are compressed by the cutting force

of the knife. In addition, interference microscopy of the cut surface of the
blocks reveals that the sections are often not flat. With Epon or Vestopal is used
as embedding medium the ridges and valleys usually do not exceed 0.5 µm in
height, i.e., 5–10 times the thickness of ordinary sections.
A small sample is taken from the specimen to be investigated. Specimens may
be from biological matter, like animal or plant tissue, or from inorganic material
such as rock, metal, magnetic tape, plastic, film, etc. The sample block is first
trimmed to create a block face 1 mm by 1 mm in size. "Thick" sections (1μm)
are taken to be looked at on an optical microscope. An area is chosen to be
sectioned for TEM and the block face is re-trimmed to a size no larger than 0.7
mm on a side. Block faces usually have a square, trapezoidal, rectangular, or
triangular shape. Finally, thin sections are cut with a glass or diamond knife
using an ultramicrotome and the sections are left floating on water that is held in
a boat or trough. The sections are then retrieved from the water surface and
mounted on a copper, nickel, gold, or other metal grid. Ideal section thickness
for transmission electron microscopy with accelerating voltages between 50kV
and 120kV is about 30–100nm.

Staining
Contrast in the electron microscope depends primarily on the differences in the
electron density of the organic molecules in the cell. Although secondary
fixation in osmium tetroxide provides some areas of electron density, this is
usually not sufficient to provide high contrast, high definition images. Staining
therefore becomes an important ingredient in enhancing contrast of areas of
interest. The efficiency of a stain is determined by the atomic weight of the stain
attached to the biological structures. Consequently, the most widely used stains
in electron microscopy are the heavy metals uranium and lead. These fall into
two major categories. Positive stains deposit electron dense material on the area
of interest on the specimen, so that it stands out as a dark area on a light
background. Negative stains penetrate and darken the interstices between areas
of interest, which then appear light on a dark background. In other words, the
stain stains the background.

Positive Stains
Positive stains are those that are used to deliberately stain the specimen of
interest. Uranyl acetate is used as a positive stain for EM. Uranyl ions react
strongly with phosphate and amino groups, staining DNA and some proteins.
Organelles composed of membranes are not stained well. Note that the starting
material is radioactive. Lead citrate may also be employed as a positive stain.
Reynolds lead citrate stain binds lead ions to negative ions, producing a general
increase in contrast. Lead is a cumulative toxin, so skin contact must be
avoided.

Negative Stains
Negative staining is most often used to highlight surface features on individual
particles, such as virions, bacteria, or cell fragments.

Appropriate staining is done on a grid and then mounted on to the TEM stage

Sample Preparation for SEM

In SEM, some samples need to be coated to make them conductive. Metals
require no preparation due to their inherent ability to conduct electricity.
However, non-metals need to be coated with a conductive material. Most often,
a thin layer of gold works. This requires the use of a sputter-coater.

Important parameters in preparing a sample for SEM imaging are as follows:

Sample Cleaning
A clean sample is essential for image clarity. For biological samples,
appropriate buffers or distilled water is used for cleaning the samples. A
surfactant is used if the sample requires more vigorous cleaning. If the
biological property of the sample is known, one might be able to use proteolytic
enzyme cleaning. To remove oils on the sample surface, appropriate solvents
are used for washing. Additionally, one can use ultrasonic baths ( Ultrasonic
cleaning is a process that uses ultrasound (usually from 20–400 kHz) and an appropriate
cleaning solvent (sometimes ordinary tap water) to clean items) for cleaning the sample.
However, ultrasonic baths require caution in order to avoid damaging the
sample.

Sample Fixation and Dehydration

Fixatives like glutaldehyde or osmium vapor can be used to maintain the
structural details of the sample. Note that if a fixative uses a phosphate based
buffer for its preparation, salt deposits may interfere with the sample’s image
quality.
Graded series of alcohol are used to dehydrate the sample and this is finished
off in the final dehydration step with 100% alcohol or acetone.

Drying
Prior to placing the sample in a high vacuum environment, it must be totally
dry. Otherwise, water vaporization will obstruct the electron beam and interfere
with image clarity. When using biological samples, one needs to be careful
when doing critical point drying (or CPD), so as to not compromise the
structural integrity of the sample. A suitable CPD instrument can help achieve
this. Alternatively, one can try using freeze drying. In this regard, freeze drying
causes the least amount of sample shrinkage in comparison to air drying or
critical point drying. However, freeze drying carries the risk of ice crystal
formation on the sample.

Specimen Coating
Specimens viewed using a conventional scanning electron microscope need to
be coated with electrically conductive material. Typically this is applied using a
sputter coater. Sputter coater's are plasma chambers with low discharge
capability that radiates a target made of heavy metal with argon atoms.
Typically, the target consists of 60% gold and 40% palladium. Gold is preferred
because it has a theory high electron output for secondary electrons, whereas
palladium is added, because it provides a more contiguous surface of metal.

Specimen Mounting
Specimens are mounted on stubs appropriate for the particular scanning electron
microscope being used. A convenient adhesive is double-stick, electrically-
conductive carbon tape. In order to assure electrical continuity of the specimen
with the stub, a small drop of silver paint may be added.