Protocol

Immersion Freezing of Cell Monolayers for Cryo-Electron Tomography

Guenter P. Resch,
1,4
Marlene Brandstetter,
1
Veronika I. Wonesch,
1,2
and Edit Urban
3
1
IMP-IMBA-GMI Electron Microscopy Facility, Institute of Molecular Biotechnology, 1030 Vienna, Austria
2
University of Applied Sciences Wiener Neustadt, 2700 Wiener Neustadt, Austria
3
Institute of Molecular Biotechnology, 1030 Vienna, Austria
INTRODUCTION
Cryo-transmission electron microscopy (cryo-TEM) is not limited to the visualization of suspended
macromolecules in their native hydrated state. It can also be used to elucidate the molecular architecture
of peripheral parts of adherent cells: After cultivation of the cells directly on EM grids, they are physically
xed by immersion freezing to yield cells embedded in thin, crystal-free layers of frozen water.
Subsequently, specimens can be visualized using cryo-electron tomography (cryo-ET). This protocol out-
lines the production of protein-coated gold particles as suitable ducial markers for electron tomogra-
phy, and how to cultivate cells on grids with a carbon support lm. It also describes how to freeze
cells to obtain optimal and reproducible results using the Leica EM GP immersion freezer, and how to
assess the results.
RELATED INFORMATION
For a brief introduction to cryo-EM, immersion freezing, the instrumentation available for this purpose,
and the Leica EM GP in particular, see Immersion Freezing of Biological Specimens: Rationale, Prin-
ciples, and Instrumentation (Resch et al. 2011a). Another protocol that describes immersion freezing of
suspended specimens, such as nonadherent cells, viruses, macromolecular complexes, and liposomes is
also available (Immersion Freezing of Suspended Particles and Cells for Cryo-Electron Microscopy
[Resch et al. 2011b]). Parts common to both protocols were duplicated so that each protocol would
be self-contained.
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institutions
Environmental Health and Safety Ofce for proper handling of equipment and hazardous materials
used in this protocol.
Reagents
Bovine serum albumin (BSA; Sigma) (5 mg/mL stock in 5 mM NaH
2
PO
4
[pH 5])
Cell culture medium
Cells of interest
Colloidal gold, 10 nm (410.011 from Aurion, Wageningen, The Netherlands; EMGC10 from BBIn-
ternational, Cardiff, UK)
Ethane gas, 99.995% (4.5; e.g., Air Liquide)
Liquid nitrogen (LN
2
), 99.95% (3.5; e.g., Air Liquide)
NaH
2
PO
4
(200 mM, pH 5)
4
Corresponding author ([email protected]).
Cite as: Cold Spring Harb Protoc; 2011; doi:10.1101/pdb.prot5643 www.cshprotocols.org
2011 Cold Spring Harbor Laboratory Press 815
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Equipment
Cell culture setup
Centrifuge (for 1.5-mL reaction tubes to be centrifuged at 40 000 g)
Cryo-grid boxes with lid, either 15- 15-mm square (Ted Pella 160-44) or 14-mm diameter round
(Leica Microsystems 16706039; Ted Pella160-40)
Each of these boxes can accommodate four grids.
Glow discharge unit, either:
Bal-Tec SCD005 sputter coater (now Leica Microsystems, Vienna, Austria) with the Au
target removed
This provides more gentle discharge; it is used for thin continuous carbon.
Instrument built according to Aebi and Pollard (1987)
This is stronger and renders the lms more hydrophilic, but should only be used for very stable lms such as
Quantifoil.
Gold (Au) 200 mesh grids with a carbon support lm with small holes (Quantifoil R 1/4, available
from Quantifoil Micro Tools GmbH [www.quantifoil.com])
Hair dryer (e.g., Steinel HG2000 fromVWR International) or Leica EMCTDdrying platform(available
from mid-2011 on)
Leica EM GP main unit (Leica Microsystems, Vienna, Austria; see Immersion Freezing of Biological
Specimens: Rationale, Principles, and Instrumentation [Resch et al. 2011a]) set up in a well-
ventilated area with low environmental humidity and good lighting
The instrument can be upgraded with a number of options:
The foot switch (16654925) makes the workow more comfortable; it takes the user to the next step in the
freezing cycle.
A binocular (viewing system; 16706433) mounted in front of the environmental chamber can be used to
closely monitor the lter paper alignment and the blotting process. In practice, however, it is not being
used much.
The forceps adjusting tool for the quick-lock forceps (16706442) and the lter paper punch (16706443)
come in very handy when using different types of forceps or lter paper (and can help to save money on
consumables).
Leica EM GP accessories:
GP quick-release forceps (set of two, well aligned to each other; one piece: Leica Microsystems
16706435)
Special forceps with insulation coating (set of two; Leica Microsystems 16701955)
Cryo-transfer container (Leica Microsystems 16706439)
Cryo-tool with M4 thread (Leica Microsystems 16701958)
Secondary cryogen liqueer (Leica Microsystems 16706438)
Filter paper for blotting, either Whatman No. 1 lter paper with precut hole (Leica Microsys-
tems 16706440) or self-made lter paper of any type with an outer diameter of 55 mm and
a central 15-mm hole, made with a lter paper punch (see above)
Light microscope slides (e.g., VWR International 631-9461)
LN
2
dewar vessels, 1-L (e.g.,VWR International)
LN
2
specimen storage system
Multiwell dishes, sterile (six- or 12-well; VWR International 734-0991 or Nunc 734-2156)
Pipette tip (200-L) or hollow needle (if Leica liqueer is not used; see Step 33)
Pressure regulator for ethane gas, 50-mbar (GDK)
Screwdrivers (or Allen key) to open/close cryo-grid boxes
Syringe
Tubing for ethane gas
Silicone tubing, 3-mm ID, 1-mm wall for the Leica liqueer
Tygon R-3603 tubing, 5-mm ID, 1.5-mm wall from Saint-Gobain/Performance Plastics for a
needle or pipette tip
Tweezers, long (300-mm; e.g., VWR International)
Vortex mixer
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METHOD
Preparation of BSA-Coated Gold Colloid
To avoid aggregation of the gold ducials on a tomography sample, use commercially available protein-coated gold (Gar-
valov et al. 2006; Lucic et al. 2007) or prepare a BSA-saturated gold colloidal stock as described here.
1. Mix 39 volumes of 10 nm colloidal gold with 1 volume of 200 mM NaH
2
PO
4
(pH 5.0). Vortex.
2. Add BSA from a stock solution of 5 mg/mL in 5 mM NaH
2
PO
4
(pH 5.0) at a dilution of 1:20 to the
buffered gold. Mix it well. Incubate it for at least 15 min at 4

C.
3. Centrifuge the gold colloid for 1 h at 40 000g at 4

C.
4. Discard the supernatant. Resuspend the red pellet in the desired buffer or medium to yield the same
volume as before centrifugation.
5. Repeat the centrifugation step.
6. Collect the nal pellet in buffer or medium (about one-tenth of the original volume) and store it at
4

C.
7. Before freezing, test the distribution and the possible aggregation of gold particles in the freshly pre-
pared stock on a glow-discharged carbon-coated grid in the TEM.
Pretreatment of EM Grids
Before cultivating cells on grids, glow discharge (plasma clean) grids in air to render the carbon support lm more
hydrophilic.
8. Place the grids to be discharged with the carbon lm side facing up onto a clean glass slide.
9. Place the glass slide into the vacuum chamber of the glowdischarge device and evacuate according
to the instructions.
10. Expose the grids for 3060 sec to the homogeneous purple discharge.
On the SCD005, use a current of 20 mA; on the homemade device (Aebi and Pollard 1987), set the high-frequency
generator to full power.
11. Vent the vacuum chamber carefully so as not to blow away the grids.
12. Use the grids as soon as possible, but in any case within 1 h.
Cultivation of Adherent Cells on Grids
13. Glow discharge Quantifoil R 1/4 200 mesh Au grids.
Copper ions are cytotoxic and nickel negatively inuences the alignment of TEMs, so do not use grids from these
materials for cell culture!
14. (Optional) Prior to plating of the cells, a matrix coating can be applied onto the carbon lm as for
glass coverslips.
15. Prepare an appropriate dilution of cells in multiwell cell culture dishes. For six-well dishes, use 3 mL of
cell suspension per well; for 12-well dishes, use 1 mL.
16. Place the grids with the carbon lm side facing up into the wells.
For ease of handling, place only one grid into each well.
17. After seeding, examine the density and the current state of the cells regularly under phase
contrast microscopy.
One or two cells per grid square on a 200-mesh grid is optimal for cryo-observation. To obtain adequately spread cells, it
may take from a few hours up to even overnight, depending on cell type.
18. Once the desired state of the cells is reached, process the grids further as described below.
Preparation of the Instrument
19. Power on the main unit.
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20. Transfer the black secondary cryogen container into its cup-shaped holder. Remove any grids that
are left inside.
It is mandatory to use this inner container in order to remove the secondary cryogen before baking out the instrument!
21. Position the transfer container holding a marked and empty cryo-grid box onto its platform.
22. Insert a newpiece of lter paper, and mount it on the magnetic holder with the metal ring provided.
Conrm the corresponding warning message on the touch screen.
23. Using a syringe, ll the water container for the humidier with 60 mL of water. Close the valve and
attach the tubing to the back side of the humidier.
24. Press the environmental parameters display to show controls. Set the chamber temperature T
C
,
humidity H
R
, and cryogen temperature to the desired values. Keep the following points in mind:
i. The chamber temperature is generally set to the temperature at which cells are cultivated.
ii. To avoid evaporation of the solvent, the humidity is typically set to 90% or above.
iii. The temperature of the secondary cryogen should be set to the lowest value that just pre-
vents solidication of the secondary cryogen (e.g., 185

C).
25. To set up the position of the lter paper relative to the grid, pick up a blank grid with one of the quick-
lock forceps that will be used for freezing. In the Load forceps position, attach the forceps to the
interlock protruding from the bottom of the environmental chamber. Go to the Setup page of
the software.
26. With the environmental chamber lowered, adjust the horizontal position of the lter paper (i.e.,
the pressure exerted onto the grid for blotting; Blotter Setting). We use a position where
the grid is bent slightly by the lter paper pressing against it. If the blot sensor (Step 31) is to
be used, this position has to be set up as well, as it is used as reference point for the sensor
measurements!
As the grid can be rotated around the vertical axis, there are two slightly different positions to set up: one with the
forceps in home position, the other one rotated by 180

. Use home position for setup.

27. Adjust the vertical position of the grid relative to the lter paper (Grid Blot Position).
We prefer a position in which the upper edge of the lter paper and the upper edge of the grid coincide.
28. If you intend to use the Transfer position for lifting the grid to the surface of the secondary cryogen
before transferring it to the grid box, set up the desired vertical position of the grid relative to the
container (e.g., just at the level of the secondary cryogen surface).
29. In Setup, Settings, set the TF heater (which produces a stream of gaseous nitrogen through the
working area to prevent contamination) to full power, unless you are planning to use the GP for a
very long time without relling the LN
2
. Increase the power of the window heater if you cannot
see the grid/lter paper contact clearly due to a fogged front window.
30. Return to the main screen. Reset the lter paper counter to 10 to have the full number of blotting
positions available.
31. Open the program list and set up/select a program suitable for freezing your specimen.
For single blot, up to 10 different programs including the parameters explained belowcan be saved. Each specimen will
require its own, optimized program. If there is any previous experience frommanual freezing a cell line, start with similar
conditions.
i. Disable rotation of the grid around the vertical axis (rst two check boxes).
This feature is not required if the grid carrying the cells is picked up in the right orientation.
ii. Activate the sensor unless it does not work with the setup of your specic experiment.
iii. Leave the option A-Plunge active to automatically plunge the specimen after blotting,
unless your thin liquid lm after blotting requires some special treatment.
iv. Set the Preblot time to 0 sec.
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v. Blot time (the time the lter paper contacts the grid for blotting liquid): Allowed values are
from 0 to 60 sec in 0.1 sec steps; 1.0 sec to 3.0 sec are good starting values for most cell
lines (see Table 1).
vi. Postblot time (an optional time after blotting but before plunging the specimen, used for
equilibration of the liquid layer [Iancu et al. 2007]).
We obtain very even ice layers without this additional wait time.
32. Cool down and ll the 1-L dewar at the base of the main unit with 2L of LN
2
. Bring the LN
2
level up to
the grid at the bottomof the working area. Wait for the vigorous bubbling of the LN
2
to stop. Fill the
transfer container holding the grid box with LN
2
and close it with its lid.
33. Condense the secondary cryogen (ethane)there are two ways to do that:
Using the Leica Liqueer
i. Connect the liqueer to the ethane bottle and place it over the secondary cryogen cup. (Con-
trary to the manual, we recommend not to cool the instrument with the liqueer in place, as
this might trap humidity in the secondary cryogen container!) The temperature reading of
the secondary cryogen container will rise. Pour LN
2
over the liqueer to speed up cooling
and wait for the temperature to equilibrate at the preset value.
ii. Fully open the main valve of the ethane bottle. Open the needle valve on the ethane bottle
slowly. After some time, white gas and subsequently liquid ethane will ll the secondary
cryogen container.
iii. Close all valves when the container is full. Do not overll; excess ethane might freeze the
liqueer onto the secondary cryogen container.
iv. Remove the liqueer carefully and place it into the small Styrofoam container provided by
Leica for safe transport.
Using a Pipette Tip (or a Needle)
i. Cut off the ne end of a 200-L pipette tip and connect it to the tubing on the ethane bottle.
ii. Wait for the temperature of the cryogen container to equilibrate to the preset value (e.g.,
185

C).
iii. Hold the pipette tip to the bottom of the secondary cryogen container and slowly open the
main and the needle valve of the ethane bottle. At the beginning, whitish gas lls the
chamber, and the sound of bubbling ethane can be used to control tip position and to
adjust the ow rate. Within 1 min, the rising level of liquid ethane is visible. Pay attention
to the temperature of the secondary cryogen container as it increases during ethane conden-
sation: If the temperature rises above 160

C, close the needle valve and wait for the cryogen

container to return to the preset temperature. Resume ethane condensation.
Table 1. Summary of freezing conditions for various cell lines
Sample Grids Pretreatment Temp. Humidity Volume Preblot Blot Postblot Ref.
B16 mouse
melanoma cells
Au 200 mesh, QF
R 1/4
Glow
discharge
37

C 90% 4 L
medium
0 sec 23 sec from
back side
0 sec
Trout
keratocytes
Au 200 mesh, QF
R 1/4
Glow
discharge
RT 90% 4 L
medium
0 sec 23 sec from
back side
0 sec Urban et al.
(2010)
CAR goldsh
broblasts
Au 200 mesh, QF
R 1/4
Glow
discharge
27

C 90% 4 L
medium
0 sec 23 sec from
back side
0 sec Urban et al.
(2010)
All specimens were blotted once with Whatman No. 1 lter paper and plunged into liquid ethane cooled to just above the
freezing point.
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iv. When the level of liquid ethane reaches the rimof the secondary cryogen container, move the
pipette tip above the ethane surface. Ethane gas ows out of the tip, replacing residual liquid
ethane that could cause burns.
v. Close the needle valve and the main valve of the ethane bottle.
34. Wait until the environmental chamber has reached the preset temperature and humidity. Keep the
working area covered, but not as tightly as to stop the gas ow.
35. Prepare another (dewar) vessel with LN
2
for relling the transfer container.
Freezing Grids
36. Bring the instrument to the Load Forceps position.
37. With the quick-release forceps, pick up the grid with the cell monolayer directly from the cell culture
dish so that cells will face away form the lter paper when blotting. Close the black slider only to the
rst notch.
38. Immediately add 4 L of prewarmed medium supplemented with ducials for cryo-ET from Step
6. To avoid dramatic changes in the osmolarity of the surrounding medium, do not use plain col-
loidal gold sol.
39. Attach the quick-release forceps to the interlock and lower the environmental chamber.
40. Press Blot (S)/A-Plunge to trigger the freezing cycle.
The blotting mechanism will be triggered.
41. Followthe blotting process closely to observe any irregularities. Check if large patches of carbon lm
are left on the lter paper.
After the postblot delay, the grid will be plunged into the secondary cryogen.
42. Once the specimen is frozen, the chamber will lift up. From now on, approach the specimen with
precooled tools only!
43. (Optional) Advance to the transfer position of the grid. This is slightly higher than the freezing pos-
ition and allows one to blot off liquid ethane with a piece of lter paper.
44. Open the lid of the transfer container with precooled forceps.
45. Disconnect the forceps fromthe grid plunger. To avoid damaging the grid at the wall of the second-
ary cryogen container, hold the forceps at the level of the black slider and tilt it out of the
beveled interlock.
46. Transfer the grid in one quick movement into the transfer container lled with LN
2
. Do not expose
the grid to the humidity of the air; do not lift it any higher than the gray ring surrounding the
working area.
47. Place the grid into the right slot of the grid box and release it from the quick-lock forceps.
48. Rell LN
2
in the transfer container, if requiredthe grid box should always be covered with LN
2
.
Keep the secondary cryogen cup covered with the plastic lid provided by the manufacturer so as
not to mix liquid ethane and LN
2
! Close the transfer container.
49. Watch out for any warning messages issued by the GP. Keep the following points in mind:
i. If necessary, rell the humidier with 20 (!) mL water.
ii. Relling LN
2
in the main dewar is only required when a warning is displayed (<25%).
iii. If you need to replace the lter paper, allow new lter paper to equilibrate with the humidity
in the environmental chamber.
This can improve the reproducibility of freezing and the evenness of the resulting ice layer.
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50. Warm up all cold and frosted tools with a hair dryer to avoid contamination of the specimen or the
cryogens with ice crystals.
Shutting Down the Instrument
51. With a precooled screwdriver or Allen key, close the grid box.
52. Rell the transfer container with LN
2
and close it.
53. Precool the handle with the M4 thread. Attach it to the specimen transfer container.
54. Lift the transfer container out of the working area and into a dewar lled with LN
2
. Then, transfer the
frozen grids either directly to the cryo-TEM or to a LN
2
storage container for later use.
55. Remove the container holding the secondary cryogen with insulated forceps and place it into the
small Styrofoam box. Allow evaporation to occur in a safe place.
56. Discard the lter paper. Leave the door of the environmental chamber open.
57. Drain the residual water from the water container.
58. Switch on a 1 h bake-out cycle.
The timer will start once the LN
2
level in the dewar has dropped to 0%.
The bake-out is not intended to sanitize the environmental chamber after freezing pathogenic specimens. Alternative
means of decontamination such as ultraviolet (UV) irradiation of the reective interior of the chamber, gas sterilization,
or wiping with ethanol have to be tested, taking into account that most components inside the environmental chamber
are not removable.
59. Once the bake-out cycle is complete (at most 1.5 h), switch off the instrument.
TROUBLESHOOTING
A number of troubleshooting issues concerning the operation of the Leica EM GP and specimen prep-
aration for cryo-TEM in general have been discussed in Immersion Freezing of Suspended Particles
and Cells for Cryo-Electron Microscopy (Resch et al. 2011b).
Problem: Cells are disrupted or deformed.
Solution: Damaged cells have been reported in a number of studies (Small et al. 2008; Lepper et al. 2009;
Urban et al. 2010), with a possible dependence on the cell line used. To minimize these problems,
remember the following:
handle the grids with extreme care
transfer the grids as quickly as possible to avoid any temperature shift and osmotic change in the
small volume on the grid
blot the cells from the back side at high humidity
Nevertheless, it is crucially important to critically assess the condition of the frozen cells (also see the
Discussion) and possibly to reconsider the choice of cell line.
Problem: Gold ducials are aggregated.
Solution: Use fresh protein-coated gold and add it right before blotting.
DISCUSSION
To localize regions of interest, we prepare low-magnication (140), minimal-dose montages of our
cryo grids using SerialEM (Mastronarde 2005) or Leginon (Suloway et al. 2005). This allows us to
assess cell density and ice thickness. An optimal result in terms of evenness of cell spreading and ice thick-
ness is shown in Figure 1A. Grid squares that have been selected at low magnication are then checked
at higher magnication (e.g., 1350) at appropriate underfocus and very low electron exposure. At
this magnication, cell bodies and even ner cell extensions accessible for tomography become visible
(Fig. 1B). Only at high magnication (>20 000) can the specimen nally be checked for successful
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vitrication, the presence of nanometer-size contamination (Immersion Freezing of Suspended
Particles and Cells for Cryo-Electron Microscopy [Resch et al. 2011b]), and the distribution of the
gold ducials.
For adherent cells, only the region at or just behind the plasma membrane is accessible for
microscopy. With some experience and underfocus, the cell boundary can be easily found (Fig. 1C),
the tilt series can be acquired at positions of interest, and tomographic reconstructions can be computed
(Fig. 1D). However, the cell has to be assessed carefully for integrity. A number of studies (Small et al.
2008; Lepper et al. 2009; Urban et al. 2010) have highlighted that cells are prone to disintegration
upon blotting/plunge freezing. This is supported by recent data from our lab in which we froze cells
grown on grids, freeze-substituted them, resin-embedded the grids, and sectioned them perpendicular
to the substrate (Wonesch and Resch, unpublished data). Although many cells were found intact and well
frozen, otherspresumably in regions with thicker iceshowed signicant ice crystal damage. Ruptured
plasma membranes, parts of cells drawn through holes, and release of cytoplasmic components into the
medium were also observed, highlighting the need for critically evaluating the condition of immersion
frozen cells from any instrument before using them for cryo-ET.
ACKNOWLEDGMENTS
We thank Ian Lamswood, Dr. Reinhard Lihl, Andreas Nowak, and Dr. Ruwin Pandithage fromLeica Micro-
systems for discussions, and Nicole Fellner for her assistance in the EMFacility. The work of G. P. R. and M.
B. was supported by the City of Vienna/Zentrum fuer Innovation und Technologie through the Spot of
Excellence grant Center of Molecular and Cellular Nanostructure. E.U. is supported by the Austrian
Science Fund (FWF project P21292-B09 to J. V. Small).
FIGURE 1. Cryo-electron micrographs and tomographic reconstructions of CAR goldfish fibroblasts grown on perforated
carbon grids and frozen according to the method outlined in this protocol. Images and zero-loss filtered tilt series were
acquired on an FEI Tecnai G2 F30 Helium at 300 kV acceleration voltage. (A) An exemplary low-magnification montage
of a major part of the grid with cells embedded in an evenly thin ice layer. (B) Medium-magnification micrograph
showing cell bodies (asterisks) and thin regions of the cells accessible for electron tomography (arrowheads). (Reprinted
from Resch et al. 2010 with permission from Elsevier 2010.) (C) High-magnification projection image of the thin periph-
eral region (lamellipodium) of a fish fibroblast imaged at low dose. (D) A 4.8-nm thick tomogram section of the lamellipo-
dium shown in C after reprojection of the tilt series. The plasma membrane and the network of long and straight actin
filaments are clearly visible (from Urban et al. [2010]). Bar (D) =250 nm (also applies to C).
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