EM Technique, TEM,transmission electron microscopy,formvar grid, electron microscopy department of TBRI, Microscopy, Support Films for Electron Microscopy Grids

1. by
Dr. Fatma Kassem
Associate Researcher of Clinical Pathology and Electron Microscopy at TBRI
2015

2.  Some basic topics about EM grid.
 Methods for grids preparation
 Preparation of solutions of plain plastic films
 Chemical precautions
 Steps of film coating grids:
◦ Collodion support films
◦ Pioloform support films
◦ Formvar coating grids
◦ Formvar grids for frozen hydrated sample
 Summarize steps of formvar coating grids

3. What is EM grid?
The EM grid is a 3.05 mm diameter, thin mesh, and it can be made
from a number of different metals (copper, gold, nickel, molybdenum,
stainless steel, tungsten, aluminum, titanium etc.), and have various
spacing patterns.
Most EM grids are made of copper because it is non-ferromagnetic
and thus minimally distorts the magnetic field of the objective lens.
The copper mesh also rapidly removes heat away from the support
film and thus prevents thermal expansion and movement of the specimen
under electron irradiation.

4.  Copper grids are usually used because they are conductive, stable in the
beam and inexpensive. But when treating the sample on the grid with
some substances such as acids, bases, some salts, etc. more expensive non-
reactive grids such as gold or platinum may be necessary.
 As The diameter of grids is standard,3.05mm. The Thickness is variable
based on the materials of the grid. The thickness range is from 10um to
25um. The mesh of the grid is defined by the number of holes within 1
inch. For example, the 200 mesh grid has 20 holes along diameter
direction, 400 mesh grid has 40 holes in diameter. With each hole size less
than 200um in 100 mesh grid, 100um in 200 mesh grid, 75um in 300 mesh
grid and 50um in 400 mesh grid.

5. The EM grid is to the TEM what the glass slide is to the light microscope. It
provides a way for supporting the specimen and delivering the specimen
into the TEM column for observation and data collection.
Grids may be used bare or, more commonly, filmed depending on the nature
of the specimen being studied.
Thin sectioned specimens, for example, are sometimes examined on bare
grids, but for highest resolution work, the sections must be mounted on
filmed grids or, better yet, on net films.
These films should be extremely thin (20 nm or less) and made of suitable
electron-transparent substance, such as plastic or evaporated carbon,
without breaking when gently handled or when irradiated with the electron
beam.

6. If high resolution work is required, plain carbon
films are the best choice because these films are
generally tough and could withstand the
bombardment of electron beam. They can also
conduct electricity and heat easily from the specimen
to the grid bars and hence reduce specimen drift due
to both charge and thermal effects.

7. Hand-made preparation Manufactured grid
Advantages:
 Age of grids is known
 Price
 Vary film thickness and
hole size to suit specimen
 Availability
Disadvantages:
 Difficult technique
 Variability
 Tim
Advantages:
 Big time saver
 Specified hole
sizes/spacings
Disadvantages:
 Expensive
 Unreliable quality
 "Freshness" issue
(hydrophobicity increases
with age)

8. Support films are generally one of three types:
1• Plain plastic such as :
Polyvinyl formal
Formvar
Vinylec (Formvar replacement)
Polyvinyl butyral
Butvar B-98
Pioloform
Nitrocellulose-based polymers (flammable and require great care in their
handling )
Collodion
Parlodion
Pyroxylin
2• Plastic coated (stabilized) with evaporated carbon.
3• Plain carbon.

9.  The grid have to be cleaned up for any dirty or chemicals before putting
specimen on it to:
 Eliminate any possibility for alternating or damaging your specimen.
 Eliminate or reduce charging problem to get quality data
 Keep electron microscope and its column clean.
(Method A):
1. Merge the grids into amyl acetate solution for several hours.
2. Drain the amyl acetate solution and rinse the grids with distiller water at least 3
times.
3. Dehydrate the grids using 100% alcohol.
4. If the grids are not clean after above procedures, put the grids into boiled 1%
NaOH solution for 5 min., rinse off the chemical using distiller water and dehydrate
with 100% alcohol.

10. (Method B):
1. Sonicating grids in acetone for 10 sec. in small beaker followed by 95%
ethanol for 10 sec.
2. Rinse with deionized water or distilled water.
3. Transfer (pour) grids onto filter paper and let dry in a dust free
environment .
(Method C):
Sonicate grids in 95% ethanol only.
(Method D):
We can skip all previous methods and use grids without any preparations if the
grids box is new and just opened.

11. 1-Formvar (polyvinyl formal) is normally used as a 0.34 % solution
in chloroform or ethylene dichloride (dichloroethane). Add 0.17g of
Formvar powder to 50 ml of chloroform and mix well on a magnetic stirrer
until dissolved.
The thickness and stability of the film can be altered by adjusting the
concentration between 0.25 % and 0.5 %.
Formvar solution is hygroscopic but will store well for several months if kept
in a brown, sealed bottle.

12. In some methods for preparing formvar grids:
The grids are initially predunked in a small volum of the formvar solution
placed faceup on a piece of filter paper and allowed to dry.
Then float formvar film on distilled water and place the predunked grids
facedown on the formvar film.
2-Pioloform ( polyvinyl butyral) is dissolved in chloroform (0.3 - 1.5
%). The solution should be stored in a refrigerator in an amber glass bottle,
and brought up to room temperature before use.

13. Formvar coated grids without a carbon layer are suitable for
TEM applications up to 100kV. The films are available on
grids made of copper, nickel or gold. Pioloform is sometimes
preferred to Formvar® because of its higher mechanical and
thermal stability, and lower bulk which can help reduce
electron scattering.

14. 3- Butvar B98 (polyvinyl butyral) is a resin containing 20 %
polyvinyl alcohol, and is normally used as a 0.25 % solution in chloroform.
It is an alternative support film to Formvar. Films made from Butvar are
hydrophilic, and therefore suitable for negative staining methods, having
good mechanical stability, high electron-transparency and minimum
intrinsic structure. Butvar B98 can also be applied to grids as an adhesive.
4- Parlodion: Its manufactured by nitrating (with nitric acid)
and then sulfating (with sulfuric acid) prepared by 0.2%
solution in isoamyle-acetate (Also used as gun powder).
5- Collodion: prepared by 2% collodion in amyl acetate solution.

15. It has been reported that one should use only amyl acetate as the solvent for
Parlodion or its sister compounds. Ethyl ether/alcohol solutions are thought to
be dangerous because if ether peroxidation has occurred, the sample could
explode upon drying).
Ethylene dichloride and chloroform are carcinogenic and methanol is toxic. Care
should be taken to avoid contact with solvents or inhaling their vapors. Old
Formvar solutions should be disposed of as hazardous waste.
Preparation of solutions must be under hood ,wearing protective coat, mask,
gloves and goggles.

16. A. Collodion support film
1. Prepare a concentration of 2% collodion in amyl acetate solution.
2. Place a drop of the collodion solution on the surface of distiller water
and allow it to spread out and dry.
3. Use tweezers to pick up the film and discard it properly. This process
will remove any dust floating on the water surface. Repeat the process
twice or more if necessary.
4. In a vibration free environment, place a drop of the collodion solution
on the surface of distiller water and allow it to spread out and dry. This
leaves a thin layer of plastic on the water surface.

17. Note: If thicker film is desired, user could add one or two more drops of
collodion drops after the first drop is well spread and dried.
Note: This method tends to produce thicker and more uneven films compared to
the formvar film.
5. Check if the film is even. Remove the film if the film appears to be uneven.
Note: A good coating film should appear an even grey to dark grey color when
viewed
by reflected light. If it appears yellowish or uneven, it is too thick and unsuitable.
6. Lay the EM grids shiny-side up (dull side down) onto the plastic film.
7. Remove the grid with film from the water using a strip of hard filter paper.
User can also use Parafilm or a clean glass slide to pick up the grids.
8. Set the grids aside in a dust free place to dry before use.

18. B- Pioloform support film:
(Pioloform produces a stronger film than formvar)
1. Prepare a concentration of 1.2% (0.3-1.5%) in chloroform
(analytical quality), adjusted with extra chloroform until films
of the desired thickness are obtained.
Note: To avoid the formation of holes in the films if the
conditions are humid, allow to dry in a lidded container thus
retaining an atmosphere of chloroform.
2. Pour approx 80ml of the pioloform solution into a 100ml
container with a tap. The container should be wide enough to
put in a glass microscope slide

19. 3. Remove dust from a glass microscope slide with tissue, and drop into the
pioloform solution. Cover the top of the container, open tap and drain
solution into the stock bottle for re-use.
4. Leave the slide to dry with the cover on, thus maintaining the chloroform
atmosphere within the container.
5. Score 2mm from the slide edge with razor, breathe on the film and lower
into a beaker of distilled water at an angle of 45°. The film should flow on
the water.

20. 6. Place acetone-cleaned grids, matt side down onto the film, separating each
by 3mm.
7. Cut a piece of 'Yellow Pages' (with text printing on both sides to allow
even uptake of water) and leave until the whole paper is wet. Remove the
grids gently from the surface with forceps.
8. For slot grids, remove from the surface of the water when 3mm has been
wetted.
9. Set the grids aside in a dust free place to dry before use.

21. C- Formvar coating grids:.
1. Prepare grids as mentioned before.
2. Polish slide and blow off any lint with compressed air. Keep the slide in a dry,
dust- free environment until use (e.g. on filter paper under an upturned beaker). If
the plastic film adheres to the slide and cannot be floated off, several different
methods of cleaning the slide should be tried as cleaning a glass slide with water
and detergent:
a) To prevent contamination with oil from the skin do not touch the slides with
bare hands (use gloves).
b) Scrub the glass slide with lint-free paper, put them into the slide holder and
soak in 2% RBS for at least one day. (RBS: mixtures of anionic and non-
ionic surfactants.
c) Rinse the slides thoroughly by running de-ionized water over the slides.
d) Dry the slides by air drying but keep them dust free or dry them in an oven
at relatively low temperature. After drying the slides can be stored in a slides
box.
 Recent attempts have shown that slides taken from a box that has been open for
several weeks or slides that have been in a petri dish for several weeks are the best.

22. 3. Submerge polished
slide 3/4 of height with
formvar solution.
4. Pour the solution into
a clean Coplin jar or
graduated cylinder.

23. 5. Drain off the formvar solution and
immediately remove slide. Touch
the edge of the slide to filter paper
to wick off the excess fluid and let
the slide stand to dry in a dust free
environment.
 The film will be milky when wet
but it will clear somewhat as it
dries( 5-10 min.).
 The thickness of the films produced
is regulated by the speed with
which the funnel drains (the longer
the slide is within the solution ,the
thicker the support film will be) and
the concentration of the formvar.

24. 6. Overfill a staining dish (or
stainless steel bowl) with distilled
water (or deionized or tap water)
and clean the surface by dragging
a kimwipe or plastic rod across the
surface to removeany floating
debris.
7. When the coated slide is dry, score
2mm from the slide edges with a
sharp, ethanol or aceton cleaned
razor blade


25. 8. Frost the slide by exhaling heavily
onto the slide to get a layer of
water vapor on and under the film.
Immediately float the films onto the
water surface by touching the
bottom of the slide, held
perpendicular or at a ~15° angle ,
to the water surface. Slowly lower
the slide into the bath, teasing the
film off as necessary. Be sure your
forceps are cleaned with ethanol.
Note: All the actions should be
gentle and the hand should
not be shaking when
lowering the slide into the
water.

26. If the film is difficult to see, a bright desk lamp
can be used to reflect light off the surface of the water
and the film.
A good film should appear an even grey to dark
grey color when viewed by reflected light. If it
appears yellowish or uneven, it is too thick and
unsuitable.

27. 9. Gently lay the grids,
shiny/bright surface
down, onto good
(uniform, gray color,
un-wrinkled, etc.)
areas of the film.

28. 10. The films with grids
are picked up by:
A- Place a small piece of clean,
white office paper onto the
surface of the grids and film and
allow it to soak up water. The
grids will show up through the
paper when the paper is fully
soaked.

29. Pick up the paper, grids
and film with forceps
and place on dry filter
paper, grid side up, in a
covered Petri dish to
dry.

30. B- Covering them with parafilm as they float on the water
surface. Pick up the edge of the parafilm and the film
with grids should come with it. If they don't, try a
different corner. Don't let your fingers touch the water
surface, or you run the risk of leaving a film in the
water and on the formvar
C- Cover slide, hard filter paper or by the slide that we
used to obtain the film covered by one side sticky
office paper

31. 11. Let the grids dry several hours
before using them. Depending on
your use, you may want to carbon
coat them to impart greater
stability to the films.
Holey films need the added
stability of thicker carbon. These
should be coated to a gray/black
color which takes about 400
milliseconds on the Emitech
carbon coater. For large particles
or complexes (>800 Å), it may be
beneficial to carbon coat very
heavily to increase the depth of
the holes.

32. 12. At this point, the grids may be examined in a light microscope with phase
contrast illumination to determine the quality of the films.
13. Carbon-Formvar films can be easily converted into carbon-only films by
placing the white paper and grids onto a piece of filter paper saturated with
ethylene dichloride. The level of ethylene dichloride should be sufficient to
completely soak the backing paper without submersing the tops of the
grids. One half hour should be sufficient time to dissolve the Formvar film
and not damage the carbon support. Remove the paper and allow it and the
grids to dry.

33. Another method for preparing pure-carbon films is to evaporate a layer
of carbon directly onto the surface of a freshly-cleaved piece of mica. The
carbon is then floated off onto water and transferred to EM grids.
14. Tape the grids and paper to a glass slide with "Scotch" tape to prevent the
grids from being jostled. Place the slide in a covered petri dish. Care must
still be used when handling the grids to prevent them from being scattered.

34. The same steps of the previous method with
following modifications:
• Add about 50 drops of a 50% glycerol/water
solution to the surface of the Formvar solution.
Place the tip of a probe sonicator onto the surface
of the solution and sonicate until mixed.
Sonication intensity should be great enough to
cause the solution to bubble vigorously. Mixing
often requires no more than about 5 seconds. The
solution should appear cloudy or milky. This
should produce numerous holes that are 1-2 µm
in diameter and suitable for use with frozen-
hydrated samples. Sonicating for longer periods
of time produces smaller holes in the film;
shorter times produce larger holes.

35.  NB:
 Grids covered with large empty Epon
sections (sections without embeded
tissue) may be used in place of
formvar for whole-mount supports.
 Other whole-mount methods describe
shorter incubation or spread time of
1-2minutes without fixatoin.

36. 1. Sonicate grids in 95% ethanol in
small beaker
2. Rinse with distilled water
3. Transfer grids onto filter paper and
let dry
4. Polish slide with lint-free cloth until
shiny/slippery, and wipe clean
5. Submerge polished slide 3/4 of
height with formvar solution(0.17 FV
powder to 50ml chloroform)
6. Drain off the formvar solution and
immediately remove slide. Let the
slide stand to dry (10 min)
7. Overfill a staining dish with distilled water
8. When the coated slide is dry, score the
edges with a sharp, ethanol-cleaned razor
blade
9. Frost the slide by exhaling heavily onto the
slide
10. Immediately float the films onto the water
surface by touching the bottom of the
slide, held perpendicular, to the water
surface. Slowly lower the slide into the
bath
11. Gently lay the grids, face down, on the
film in the regions that are of the proper
thickness and without wrinkles etc
13. The films with grids are picked up by
cover slide
14 Let the grids dry several hours before
using them. Depending on your use, you
may want to carbon coat them to impart
greater stability to the films.

37. • After picking up the formvar grids from DW:
1. let them completely dry.
2. place the paper with the film and grids onto a piece of filter paper that
is just saturated with methanol. The glycerol in the Formvar solution
often produces bubbles in the film not open holes. The methanol
treatment will break the thin film over these "pseudo-holes".
3. Soak in a covered petri dish for about 40-50 minutes.
4. Allow the film to dry before carbon coating.