Electron Microscopy (EM) Grid
and Carbon film Preparation for
Cryo-Electron Microscopy
Cryo-EM tutorial
July25~29, 2005
Zongli Li
What I will talk about……

Some basic topics about EM grid

Home-made grid preparation

Grid cleaning

Carbon coating and carbon film
preparation
Some basic topics about EM grid
What is EM grid?
 The EM grid is a 3.05 mm diameter, thin-foil 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.
 The diameter of grids is standard, as 3.05mm. The Thickness is
variable based on the materials of the grid. The tickness 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.
 Since the diameter of grid is a standard length, 3.05mm, each hole
size is less than 200um in 100 mesh grid, 100um in 200 mesh grid,
75um in 300 mesh grid and 50um in 400 mesh grid.
 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.
Some basic topics about EM grid (Contd.)
What we need to do with EM grid
before we can use it?
 Often an electron lucent film is deposited upon the grid to aid in
specimen support. The TEM techniques that are most often used to
investigate macromolecular structures require films made of a thin
layer of carbon which may or may not have numerous small
perforations. The macromolecules are then examined while adhered
to the carbon or while suspended in the holes.
 The grid have to be cleaned up for any dirty or chemicals left over
from manufacture or process of depositing electron transparent film
before putting specimen on it.



Eliminate any possibility for alternating or damaging your specimen.
Eliminate or reduce charging problem to get quality data
Keep OLA, pole piece and column clean.
Some basic concepts about EM grid (Contd.)
How to chose right gridsome preliminary considerations




The type of grids and films used are usually determined by the desired
specimen preparation technique and sample characteristics.
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.
Both holey films and continue thin carbon films are mounted on grids with
appropriate mesh value for stability in the beam at higher magnifications.
Vitrified samples are best examined when suspended in the holes of a
holey carbon film, while negative stained samples are examined on
continuous carbon films.
Some basic topics about EM grid (Contd.)
Home-made grid Vs. commercially available grid
(http://www.grid-tech.com/product.htm
http://www.quantifoil.com/)
• Manufactured holey grid

Advantages:
• Big time saver
• Specified hole sizes/spacings

Disadvantages:
• Expensive
• Unreliable quality
• "Freshness" issue (hydrophobicity increases with age)
• In house preparation

Advantages:
•
•
•
•

Age of grids is known
Price
Vary film thickness and hole size to suit specimen
Availability
Disadvantages:
• Difficult technique
• Variability
• Time
Home-made grid preparation*
Grid with continuous thin c-film

•
•
•
Evaporate a uniform carbon layer over plastic films or freshly
cleaved mica to be used as a stand alone support film
Produce very thin support films (~250Å) for use in testing
microscope or negative staining
Evaporate a thin carbon layer over holey films for use in CryoEM
grid with holey c-film

•
•
•
Provide a secondary mesh over the copper grid for the suspension
of a thin film of vitrified ice forming an unsupported layer within the
holes of that mesh
Produce holes within that mesh that have appropriate diameters and
depth (depth = film thickness) to contain the correct amount
specimen solution for freezing to the appropriate ice thickness
Produce a support that has the necessary surface characteristics to
allow for the quick freezing of thin films of hydrated specimens
*: Some pictures and materials are borrowed from web site
http://bilbo.bio.purdue.edu/~baker/documentation/sample_and_prep/b2.htm for this section
Home-made grid preparation
--grid with continuous thin c-film (1)
1) Pre-clean copper grids.
a) Copper grids should be pre-cleaned
by sonicating for 10 sec. in
Acetone, followed by 10 sec.
sonication in ethyl alcohol.
b) Allow grids to dry on filter paper in
a dust-free environment before
use.
2) Prepare Formvar solution.
a) Add 0.12g of Formvar powder to
50 ml of ethylene dichloride and
mix well on a magnetic stirrer until
dissolved.
b) Pour the solution into a clean
Coplin jar. This solution is
hygroscopic but will store well for
several months if kept in the dark,
tightly sealed, and refrigerated.
Home-made grid preparation
--grid with continuous thin c-film (2)
3) Clean 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,
http://www.chromspec.com/Catalogue/Misc/CleaningSolutions.html)
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.
4) Coat the clean slide with Formvar.
a) Dip the cleaned slide into the Formvar solution
and touch edge to filter paper to drain off the
excess fluid. The film will be somewhat milky
when wet but it will clear as it dries.
b) Dry upright in a dust-free environment (this
requires 5 to 10 min.
Home-made grid preparation
--grid with continuous thin c-film (3)
5) Remove Formvar film from
slide and add grids.
a) Fill the black dish with deionized water until a
meniscus is formed on top of
the dish. Then drag the
lucite rod across the surface
of the water to remove any
floating debris.
b) Cut the edges of the
Formvar film with an
acetone-cleaned razor blade
Home-made grid preparation
--grid with continuous thin c-film (4)
c) Thoroughly breathe over the length
of the slide to loosen the film, and
float the film off onto a clean water
surface by slowly immersing the
slide into the water at a ~25° angle.
If the film is difficult to see, a bright
desk lamp can be used to reflect
light off the surface of the water and
film.
d) Place grids, shiny/bright surface down,
onto good (uniform, gray color, unwrinkled, etc.) areas of the film.
Home-made grid preparation
--grid with continuous thin c-film (5)
e)
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.
f)
Pick up the paper, grids and film
with forceps and place on filter
paper grid side up in a covered
Petri dish to dry.
Home-made grid preparation
--grid with continuous thin c-film (6)
6) Carbon coat film to desired thickness.
7. Remove Formvar film.
a) Place the paper with grids onto a piece of filter paper saturated with
ethylene dichloride in a covered Petri dish. 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.
b) Remove paper with the grids and let dry in a dust-free area.
Home-made grid preparation
--grid with holey c-film (1)
1) Pre-clean copper grids.
2) Prepare Triafol (cellulose acetate butyrate, Fisher Scientific,
https://www1.fishersci.com/Coupon?cid=1334&gid=84054) solution.
a) The night before making holey grid films, prepare water and ethyl
acetate solution. Take a 200 ml glass bottle which can be used with
organic solvents. Add 5 mls of de-ionized water and then 40 mls of
ethyl acetate, shake well and then allow the solution sit at room
temperature over night.
note: Do not use the solution for more than a week or two.
b) The concentration of Triafol solution will depend on the size of the hole
you want. We can use 0.2% Triafol solution to make the film with hole
size around 2 µm.
c) The Triafol is dissolved in the ethyl acetate first and then the hydrated
ethyl acetate is added (1:1 in volume). Store the solution in a Coplin
jar with a cover at room temperature. The solution should be on ice at
lease 30 to 45 min before making holey film. Do not use the solution
for more than 2 or 3 days.
Home-made grid preparation
--grid with holey c-film (2)
3) Clean 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.
c) Rinse the slides thoroughly by running de-ionized water over the slides
and soak them in 0.03% Amiet for at least one day.
d) After the Amiet treatment, rinse the slides with de-ionized water as
follows: dip the slides in a dish of de-ionized water and take them out.
Change the water and dip the slides again. Change the water again and
then dip the slides but when you remove them this time pull the slide
holder out slowly so that there is no trace of water on the slides except
near the edges.
e) 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.
Home-made grid preparation
--grid with holey c-film (3)
4) Making holey film.
a) Set-up for making holey film.
i. Find a small room that can be humidified without interruption.
Humidify the room at least 3 hours before making the holey film.
ii. Keep Triafol solution (that you prepared and put on ice) in the room.
iii. Metal plates pre-cooled in refrigerator are put on ice chest and used
to cool the slide below the dew-point.
b) Making holey film.
i. Take a clean, Amiet treated slide and place on metal plate. Do
several slides, each with different time (15 seconds to 1 min). Find
the time which give you best results.
ii. After slide is cooled, take out and wave it in path of humidifier spray.
Immediately dip it into the Triafol solution. When the slide get into
the solution as far as it can, quickly pull it out and dry it by leaning it
against the ice bucket with one end touching ice.
iii. Check slides on phase-contrast microscope see if the results is
satisfied.
iv. Store good slides in slide box until ready to make holey grids.
Home-made grid preparation
--grid with holey c-film (4)
5) Preparing holey grid.
a) Prepare 0.5% Pelex (anionic surfactant) solution. This solution can be
kept in refrigerator indefinitely.
b) Fill a Coplin jar with 0.5% Pelex solution and place the slide with holey
film in the solution. Let slide soak for about 3 hours. Keep slides
separate if more than one slide have been put into the solution.
c) Take the slides out and let it air dry.
d) Fill the black dish with de-ionized water until a meniscus is formed on
top of the dish. Then drag the lucite rod across the surface of the water
to remove any floating debris.
e) Cut the edges of the slide with film with an acetone-cleaned razor blade.
Float the film off onto a clean water surface by slowly immersing the
slide into the water at a ~15° angle. If the film is difficult to see, a bright
desk lamp can be used to reflect light off the surface of the water and
film.
f) Place grids, shiny/bright surface down, onto good areas of the film.
g) 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.
Home-made grid preparation
--grid with holey c-film (5)
h) Pick up the paper, grids and film with forceps and place on filter paper
grid side up in a covered Petri dish to dry.
i) After drying overnight, pick grids off the paper with tweezers and put
them on clean glass slide with film side up.
j) Carbon coat film to desired thickness. (a thick carbon layer compared to
continuous c-film).
k) Remove plastic film. There are many different ways to remove plastic film, you
should find the way which works best for your experiment.her
l) After the plastic film removing, place the grids on another clean glass slide with the
carbon side down. Carbon coat the other side and store them in clean grid box for
future use.
Home-made grid preparation
--grid with holey c-film (method 2) (1)
1) Pre-clean copper grids.
a) Copper grids should be pre-cleaned by sonicating for 10 sec. in acetone,
followed by 10 sec. sonication in ethyl alcohol.
b) Allow grids to dry on filter paper in a dust-free environment before use.
2) Prepare Formvar/Chloroform solution.
a) Add 0.17g of Formvar powder to 50 ml of chloroform (Do not use ethylene
dichloride) and mix well on a magnetic stirrer until dissolved.
b) Pour the solution into a clean Coplin jar. This solution is hygroscopic but
will store well for several months if kept in the dark, tightly sealed, and
refrigerated.
3)
Clean a glass slide with water and detergent.
Home-made grid preparation
--grid with holey c-film (method 2) (2)
4) Produce a suspension of glycerol droplets
in the suspension.
a) Add about 50 drops of a 50%
glycerol/water solution to the surface
of the Formvar solution.
b) 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 violently.
This often requires no more than
about 5 seconds. The solution should
now 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.
Home-made grid preparation
--grid with holey c-film (method 2) (3)
5) Coat the clean slide with Formvar.
a) Immediately after sonicating, dip the cleaned slide into the Formvar
solution and touch edge to filter paper to drain off the excess fluid. The
film will be somewhat milky when wet but it will clear somewhat as it
dries.
b) Dry upright in a dust-free environment
6) Remove holey Formvar film from slide and add grids. (Same as before)
7) Let the film dry and perforate the pseudo holes.
a) When the paper is completely dry, place the paper with the film and
grids onto a piece of filter paper that is just saturated with methanol.
Soak in a covered petri dish for about 40-50 minutes. This should
perforate any pseudo-holes in the films.
b) Let them dry completely before carbon coating.
8) Carbon coat film to desired thickness.
Home-made grid preparation
--grid with holey c-film (method 2) (4)
9)
At this point, the grids may be examined in a light microscope with phase
contrast illumination to determine the quality of the films.
10) Remove Formvar film.
Grids Cleaning
Why need to clean grid before use?
• Good for your specimen
•
Good for imaging
•
Good for microscope
Organic Solvents used for grid cleaning
Two organic solvents have been used in this lab,
• Acetone ((CH3)2CO )
• ethyl acetate (CH3COOC2H5 )
People in other places also use chloroform (CHCl3) and
ethylene dichloride (C2H4Cl2)
Caution:
Ethylene dichloride, chloroform are carcinogenic, ethyl acetate and methanol
are toxic. Care should be taken to avoid contact with these solvents or inhaling
their vapors. Although acetone is less toxic, breathing moderate- to-high
levels of acetone for short periods of time, can also cause nose, throat, lung,
and eye irritation and other health problems.
Diagram for cleaning gridsHome-made holey grid (1)
Diagram for cleaning gridsHome-made holey grid (2)
Quantifoil grid
In the final stage of the production process
of QUANTIFOIL® holey film, carbon is
evaporated onto a plastic holey film, and
the plastic is dissolved. The grids are
washed with chloroform, ethyl acetate and
acetone. However, not all plastic can be
removed. Therefore, the final thickness of
the foil is about 20 nm, although only 10
nm of carbon is evaporated onto the
plastic.
R1.2/1.3 quantifoil grid
Diagram for cleaning gridsQuantifoil grid
Acetone
water
water
Carbon coating and
carbon film preparation
• Evaporate a uniform carbon layer over plastic films
or freshly cleaved mica to be used as a stand alone
support film
• Produce very thin support films (~250Å) for use in
testing EM and negative staining
• Evaporate a slightly thicker carbon layer over holey
films for use in CryoEM
Operation of carbon evaporator
EMitech K950 & K950X
The evaporator has a small vacuum chamber and
is very sensitive to oil contamination. To keep it
oil-free the following rules are necessary:
•Never work inside the chamber without gloves.
•Never use solvents to clean inside the chamber. Use a brush to
remove the carbon flakes from the lid, the stage, and from the
walls.
•Never touch anything that goes inside the chamber with bare
hands. Use tweezers whenever possible.
•Never use solvents or vacuum grease on the bell jar gaskets;
they become sticky. We use talc powder on these gaskets to
prevent stickiness.
Carbon evaporator
EMitech K950
EMitech K950X
Procedure
for operating the carbon evaporator (1)
1) Put on gloves!
2) Turn the power switch on
(in back of the unit on the
right side above the power
cord). The "Stop" button
should be lit . The unit
should be at atmospheric
pressure. Open the valve on
the regulator of nitrogen
cylinder. This will supply dry
nitrogen to vent the
chamber when you need to
stop the evaporator.
Procedure
for operating the carbon evaporator (2)
3) Open the bell jar lid and
check the carbon rods.
The sharpened tip should
be at least 3-4 mm long
and make good contact
with the broad tip of the
other rod. If you need to
clean the lid and rod
holders, cover the upper
surface of the jar with
aluminum foil and clean
the lid with a small
brush.
Procedure
for operating the carbon evaporator (3)
4) Put your grids or mica in the slot on the stage along with a marker (A piece of
filter paper works just fine). Ensure that nothing protrudes beyond the edge of
the stage as the venting process can cause any such object (i.e. grids) to fly all
over the chamber. Lower the lid and check to make sure that it is firmly seated
and properly closed.
5) If you need to change parameters (most of the time the standard parameters
are fine), push the "Enter" button and follow the instructions; otherwise push
the "Start" button (Fig. 1) and wait until the unit is ready. The rotary pump
should start immediately and you should hear an accelerating whine which is
the turbomolecular pump. On the display is a setting that shows the actual
vacuum in the chamber and the speed of the turbomolecular pump (displayed
in percent of maximum speed). If the pump accelerates smoothly that is a
good indication that the unit is working properly. In approximately 3 minutes
the speed should reach 100%. Wait until the gauge shows that the vacuum is
at the "high vacuum" level and the initial pumping cycle is complete. Currently
this cycle is set at 5 minutes (changeable on the parameters page).
Procedure
for operating the carbon evaporator (4)
7) When the initial pumping is done the display should show "Push up arrow to
outgas or down to evaporate. Stop to escape". The "Stop" button is used to
escape from the previous menu or to stop the current process. Outgassing of
the rods is necessary if they have not been used before, otherwise you may
proceed with evaporation. In the outgas mode start with the current knob
turned completely to the CCW position and slowly increase the current by
rotating the knob CW. Watch the rods and continue to increase the current
until the sharpened rod begins to glow. Allow the rods to glow for several
seconds and then stop the outgassing by pushing the "Stop" button. Return
the knob to zero (fully CCW).
8) You will then again get the message "Push up arrow to outgas or down to
evaporate. Stop to escape". Enter the evaporation mode by pushing the
"Down" arrow button. The stage will begin rotating. Slowly increase the
current by rotating the knob CW until you are satisfied with the evaporation
rate. You may want to watch the "demand" or "actual value" on the display to
decide where to start the next time. Sparking rods will coat the grids much
quicker than a slow, glowing evaporation but the carbon will have a much
more inconsistent granularity.
Procedure
for operating the carbon evaporator (5)
8) When you are satisfied with the film thickness (Fig. 5) stop the evaporation
by pushing the "Stop" button. If you find that the film is not dark enough,
go back to evaporation mode and put more carbon onto the grids. You can
repeat this as many times as you want while the carbon rods remain in
contact.
9) To remove the grids (or mica) push the "Stop" button. The display will show
"Slowing down turbo" followed by "venting chamber". We use a pulsed
venting process to return the chamber to atmospheric pressure, meaning
that air is admitted to the chamber in small bursts, to help prevent grids
from flying off the stage. In about 30 seconds to 1 minute the chamber
should be at atmospheric pressure. If it is not at atmosphere, press the
"Stop" button again to allow more air into the chamber. Open the lid and
remove the bell jar to get the grids. Replace the bell jar and close the lid.
Never leave the unit under vacuum unless the turbomolecular pump is
running to prevent back streaming oil vapor into the chamber.
10)Turn off the power on main unit and close the nitrogen valves. The bell jar
should be cleaned when it becomes dark. If after your use the jar appears
dark, please clean it as a courtesy to the next person.
Procedure
for operating the carbon evaporator (6)
Cleaning the Bell Jar:
1) Carefully remove the bell jar by pulling it straight up and past the implosion shield. Be
extremely careful when handling the bell jar because it is expensive. Remove both
gaskets.
2) Soak the bell jar with soapy water for a couple of minutes then gently rub it with
paper towels. When the jar is clean, rinse it with deionized water and wipe it dry with
Kimwipes.
3) Apply a thin coating of Bell Bright to the inner surface of the jar. There should not be
any liquid droplets on the surface. Let it dry for a couple of minutes and then replace the
gaskets. Do not forget to use Bell Bright or the carbon will be nearly impossible to
remove the next time it is cleaned.
Carbon coating grid with holey or continuous
plastic film
To get holey carbon grids or grids with continuous
carbon film.
Put more carbon on holey carbon grid
There are different reasons for putting more carbon
on holey carbon grid
• Increase the strength of the holey carbon grid so that the
holey carbon film will not be damaged during washing
• Increase the thickness of holey carbon film to meet the
freezing different specimens.
• Change the hydrophilicity of the grid so that one can get idea
ice thickness and particle distribution.
Making the stand-along thin carbon film using
freshly cleaved mica
Purpose
• Make the continuous c-film grid for testing EM
• Make the continuous c-film grid for negative staining
• Make the continuous c-film grid for some special samples
Procedure
• Cleave mica to get the fresh, flat surface where the c-film will be made
• Coating the freshly cleaved mica with fine, continuous thin c-film
Put the carbon film on top of the holey carbon
grid or bare grid
• Cut a small piece of mica that have thin c-film on it (cut around off by
1 mm.
• Float the c-film off onto the surface of water
• Cut Whatman #1 filter paper to small pieces (4 mm x 4mm)
• Put the grid onto the a piece of filter paper with shinny side up. Use
tweezers to take the grid and filter paper together and pick up a piece
of c-film from underneath. Put them on filter paper and let them dry
completely in dust free environment. Store them in grid box for future
use.