C
H
A
P
T
E
R
28
Correlative Light and Electron
Microscopy of the Cytoskeleton
Tatyana M. Svitkina and Gary G. Borisy
effort in a single cell. If the efficiency of recovering a
cell for EM is low, the investment is lost.
For studying cytoskeletal components, we have
chosen detergent extraction-chemical fixation-critical
point drying (CPD)-TEM of platinum replicas as a
basic procedure (Svitkina et al., 1995) because it allows
a higher yield of successful results in comparison
with alternative approaches. In the replica EM
technique, the contrast is created by shadowing of
three-dimensional (3D) samples with metal. The
purpose of detergent extraction is to uncover the
cytoskeleton and make it available to metal coating,
yet to preserve it in its entirety as in the living state
(Lindroth et al., 1992). The composition of the extraction solution is designed to achieve this goal. Chemical fixation provides cell structures with physical
resistance against subsequent harsh procedures. Our
fixation procedure includes consecutive treatment
with glutaraldehyde, tannic acid, and uranyl acetate.
Drying exposes surfaces of the specimen for vacuum
shadowing. The preservation of 3D structure is the
major concern during EM processing, especially
during drying. The main source of problems is the
surface tension at the liquid-gas interface, which will
crush fragile cytoskeletal structures if the interface
passes through the sample. CPD is a simple and reliable technique, which circumvents this problem and
preserves the complicated 3D structure of the
cytoskeleton (Ris, 1985).
This article describes the procedure for preparation
of cells for correlative EM after light microscopic
observation, as well as the combination of this
approach with immunostaining. Direct comparison of
I. I N T R O D U C T I O N
Light and electron microscopy (EM) each have
certain advantages and limitations for the investigation of the cytoskeleton. Light microscopy allows for
kinetic observations in living cells; in particular,
modern fluorescence technology affords imaging of
single fluorophores with high temporal resolution.
However, the spatial resolution of light microscopy is
limited to approximately 200-300nm. In contrast, EM
affords high spatial resolution but provides only static
images and is not applicable to living cells. Correlative
light and EM is a way to combine the advantages
of these two techniques and link cell structure and
dynamics. The main strategy of this approach is to
follow the dynamics of a living cell by time-lapse
imaging and subsequently analyze the same cell
by EM.
The success of correlative microscopy imposes
special demands at both the light and the EM level. To
allow for precise identification of corresponding features in EM, light microscopy should be performed at
the highest possible resolution and allow for the fast
cessation of dynamic cellular processes at the end of
the light microscopic observation. For fluorescence
light microscopy, issues of photodamage and phototoxicity become more critical, as EM is able to reveal
damage not recognizable at the light microscopic level.
The key requirements for the EM procedure are
quality, reproducibility, and yield. Yield is essential
because detailed observation of individual living cells
places a high investment of investigator time and
Cell Biology
277
Copyright 2006, Elsevier Science (USA).
All rights reserved.
2 78
ELECTRONMICROSCOPY
living cells and platinum replicas of their cytoskeletons using a number of different markers demonstrated that our protocol does not introduce alterations
in the distribution of several cytoskeletal elements
(Svitkina et al., 1997; Svitkina and Borisy, 1998,
1999).
II. M A T E R I A L S A N D
INSTRUMENTATION
1. Leibovitz's L-15 medium (Cat. No. 21083-027,
GIBCO)
2. Phosphate-buffered saline (PBS) (Cat. No. 21040-CV, Cellgro)
3. PIPES (Cat. No. 528131, Calbiochem)
4. Triton X-100 (Surfact-Amps X-100, Cat. No.
28314, Pierce)
5. Polyethelene glycol (PEG), MW 40,000 (Cat. No.
33139, Serva Electrophoresis) or MW 35,000 (Cat. No.
81310, Fluka)
6. Taxol (paclitaxel) (Cat. No. T7402, Sigma)
7. Phalloidin (Cat. No. P2141, Sigma)
8. Glutaraldehyde (Cat. No. 01909-10, Polysciences)
9. Sodium cacodylate (Cat. No. C-4945, Sigma)
10. Tannic acid (Cat. No. 1764, Mallinckrodt)
11. Uranyl acetate (J. T. Baker Chemical Co.)
12. NaBH4 (Cat. No. 213462, Aldrich)
13. Bovine serum albumin (BSA) (Cat. No. A-7906,
Sigma)
14. Tween 20 (Cat. No. X251-7, J. T. Baker Chemical
Co.)
15. Tris (Trizma-HC1) (Cat. No. T-3253, Sigma)
16. Gold-conjugated antibodies (Cat. Nos. G-7777,
G-3779, G-5527, G-5652, Sigma; Cat. Nos. 115-215-068,
111-215-144, Jackson Immunoresearch Laboratories)
17. Ethanol (Cat. No. 15055, Electron Microscopy
Sciences)
18. Molecular sieves (4A, 8-12 mesh) (Cat. No.
M514-500, Fisher)
19. Hydrofluoric acid (HF) (Cat. No. A147-1, Fisher)
20. 35-mm tissue culture dishes
21. 22 x 22-mm glass coverslips (No. 1.5)
22. Silicon vacuum grease (Dow Corning)
23. Gold wire (Cat. No. 21-10, Ted Pella)
24. Platinum wire (Cat. No. 23-10, Ted Pella)
25. Tungsten wire (Cat. No. 27-3-20, Ted Pella)
26. Carbon rods (Cat. No. 61-13, Ted Pella)
27. Platinum loop
28. Diamond pencil
29. Double-sided tape (Scotch)
30. Lens tissue (Kodak)
31. Post-It notes
32. Electron microscopic grids (e.g., Cat. No. G50,
Ted Pella)
33. Locator grids (e.g., 7GC200, Ted Pella)
34. Holders for critical point drying. We use a
homemade holder (Fig. 1), which consists of a wire
basket that fits the size of the critical point dryer's
chamber. Such a design is good for correlative EM, as
it is not very demanding to the shape and size of the
coverslips. For noncorrelative EM, commercially
available holders can be used (e.g., Cat. No. 8762 for
coverslips under 7 m m or Cat. No. 8766 for round 12m m coverslips, Tousimis).
35. 50-ml glass beakers
36. Homemade scaffolds (Fig. 1)
37. Stirrer bars (Fig. 1)
38. Fine tip forceps
39. Dissection microscope
40. Critical point dryer. We use a semiautomatic
Samdri-795 or manual Samdri PVT-3 (Tousimis). Other
devices have also been used successfully. As a source
of liquid CO2, we use high-quality carbon dioxide (Cat.
No. CD 4.8SE, Praxair); however, lower quality grades
can also be used if they have low water and carbohydrate contamination. The cylinder should be equipped
FIGURE 1 Accessoriesfor CPD: specimen holder, lid for holder,
and scaffold are made from stainless steel mesh. Inset shows an
assembled set.
CORRELATIVE LIGHT AND EM STUDIES OF CYTOSKELETAL DYNAMICS
with water and oil absorbing filter (Cat. No. 8781/82A,
Tousimis).
41. Vacuum evaporator. We use an Edwards 12E1
evaporator equipped with rotary and diffusion
pumps, power supply, rotary stage, and thickness
monitor (Cat. No. QM-311, Kronos, Inc.). Newer
models with suitable configuration are now available
from Edwards and other sources.
279
gold coating may vary. It should be clearly visible
by eye as a purple transparent deposit. Avoid too
thick of a coating because the gold may then contaminate the clear glass area under the grid.
3. Remove grids, collect coverslips, and bake them at
160~ overnight. Baking prevents dislocation of
gold grains by cultured cells.
2. Cultivation chambers
III. P R O C E D U R E S
A. Cell Culture and Light Microscopy
Details of cell cultivation, introduction of fluorescent probes into cells, and light microscopic observation are beyond the scope of the present description.
However, certain issues are specific to correlative
microscopy and these we discuss.
1. Preparation of Locator Coverslips
Locator coverslips are helpful in facilitating the relocalization of the same cells. The reference marks on the
coverslip should be recognizable by both light and EM.
We use glass coverslips coated with a thin layer of gold
through a locator grid (Fig. 2). Cells are selected within
clear uncoated glass areas corresponding to the solid
parts of the locator grid.
Steps
1. Put one or two locator grids in the center of 22 x 22mm glass coverslip. Place coverslips onto the stage
of vacuum evaporator.
2. Evaporate gold onto coverslips using a procedure
suitable for the particular evaporator. Thickness of
The locator coverslips may be mounted into different types of chambers suitable for cell cultivation and
observation. For correlative microscopy, the chamber
design should allow for the fast exchange of media. In
our laboratory, we typically mount coverslips onto the
hole in the bottom of 35-mm tissue culture dishes (Fig.
2) and perform light microscopic observations in open
dishes. Compared to any kind of sealed chambers, this
design allows faster processing for EM and decreases
the lapse between light and EM observations. To
prevent pH shift in the medium during observation,
we use Leibovitz's L-15 medium.
Steps
1. Smooth edges of the hole before mounting the
coverslip.
2. Apply a thin line of vacuum grease along the edges
of the hole inside the dish. Use the minimum
amount of grease required to prevent leakage to
avoid complications during subsequent excision of
the central area of the coverslip with the desired
ceils (see later).
3. Mount the coverslip with gold-coated side facing
upward. Press firmly along the line of grease until
grease forms a continuous clear circle around the
hole without any air bubbles, which may cause
FIGURE 2 Locatorcoverslip for relocalization of cells. (a) A 22 x 22-mm coverslip was coated with gold
through the locator grid and mounted over the 18-mm hole in a 35-mm plastic dish with vacuum grease.
(b) Diagram showing gold pattern on the coverslip. (c) Xenopus epidermal keratocytes growing on a coverslip with gold pattern. The imaged area corresponds to the box in b. Dark squares at left are gold islands
corresponding to holes in the locator grid, which was used for shadowing.
280
ELECTRON MICROSCOPY
leakage. Sterilize with UV irradiation before plating
cells.
B. Preparation of Cytoskeletons
1. Extraction and Fixation
Solutions
1. PEM buffer: 100mM PIPES, pH 6.9; I mM MgC12;
and I mM EGTA. To make 100 ml of 2x stock solution,
mix ~70ml of distilled water and 6g of PIPES. While
stirring, add concentrated KOH to this turbid solution
until it almost clears. Add 76 mg of EGTA and 200 btl of
1M stock of MgCI2, adjust pH to 6.9 with 1N KOH,
and complete with distilled water until 100ml. Store
at 4~
2. Extraction solution: 1% Triton X-100, 4% PEG in
PEM buffer supplemented (optionally) with 2 btM taxol
and / or 2 btM phalloidin. To make 10 ml, combine 5 ml
of 2x PEM, I ml of 10% Triton X-100, 400mg PEG, and
complete to 10ml with distilled water. Stir for ~1015min until dissolved. Store at 4~ and use within 1
week. Add 10btl of 2mM taxol (paclitaxel) in dimethyl
sulfoxide (DMSO) or 10btl of 2mM phalloidin in
DMSO before use.
3. Sodium cacodylate stock: 0.2M Na-cacodylate, pH
7.3. Dissolve 4.28 g of Na-cacodylate in distilled water,
adjust pH to 7.3 with HC1, and complete until 100ml.
Store at 4~
4. Glutaraldehyde: 2% glutaraldehyde in 0.1M
sodium cacodylate, pH 7.3. To make 10ml, combine
5 ml of 0.2M Na-cacodylate, 0.8ml of 25% glutaraldehyde, and 4.2 ml of distilled water. Store at 4~ and use
within a week.
5. Tannic acid: 0.1% aqueous tannic acid. Weigh
10mg of tannic acid and dissolve in 10ml of distilled
water. Use within a day.
6. Uranyl acetate: 0.1% aqueous uranyl acetate.
Weigh 10mg of uranyl acetate and dissolve in 10ml of
distilled water. Remove undissolved salt by centrifugation. Store at room temperature.
Steps
1. Using a pipette or vacuum aspirator, aspirate
culture medium from a dish while it is on the microscope stage. Immediately, but gently, add prewarmed
to 37~ PBS with a wide-mouth pipette or pour from
a beaker.
2. Aspirate PBS and immediately add extraction
solution at room temperature. Exchange of media
should be fast to avoid cell damage by drying
and to decrease the lapse between living and lysed
state of the cell. Incubate for 3-5min at room
temperature.
3. Rinse cells with PEM buffer at room temperature
two or three times, I min each.
4. Add glutaraldehyde and incubate for at least 20
min at room temperature. If necessary, specimens can
be refrigerated at this stage and stored for several days
in sealed dishes to prevent drying. Before further processing, specimens should be brought back to the room
temperature. If immunogold staining is required, it is
best to do it after this step (see later).
5. Remove glutaraldehyde and add tannic acid. No
washing is necessary before application of tannic acid,
although it is not contraindicated. Incubate for 20min
at room temperature, rinse in three changes of distilled
water, and incubate for 5min in the last change of
water.
6. Remove water and add uranyl acetate; incubate
for 20 min at room temperature. Replace uranyl acetate
with distilled water.
2. Immunostaining
Platinum replica EM is compatible with immunoelectron cytochemistry and with the use of colloidal
gold as an electron-dense marker. The difference in
electron density between colloidal gold particles and
the platinum layer is sufficient for detection of the
immune reaction in coated specimens. The specific
protocol for immunogold EM depends on the primary
antibody and its ability to recognize antigen under
particular conditions. Initial evaluation of the quality
of staining at the light microscopic level is strongly
recommended. For most antibodies we use immunostaining after glutaraldehyde fixation because it
provides the best structural preservation. We do not
recommend using formaldehyde or methanol fixation,
as they are generally inadequate for preserving structure at the EM level.
Solutions
1. Sodium borohydrate NaBH4: 2 m g / m l NaBH4 in PBS.
Weigh 20mg of NaBH4 and complete with 10ml of
PBS. Use immediately.
2. Primary antibody: The required antibody concentration should be estimated in preliminary light microscopic experiments. For EM, use the antibody
concentration that produces a bright immunofluorescence signal.
3. Buffer A: 20mM Tris-HC1, pH 8.0, 0.5M NaC1, and
0.05% Tween 20. To make 100ml of 5x stock solution, dissolve 1.2g Trizma-HC1 and 14.5g NaC1
in distilled water. Adjust pH to 8.0. Add 250btl of
Tween 20 and complete to 100ml with distilled
water. Store at 4~
CORRELATIVE LIGHT AND EM STUDIES OF CYTOSKELETAL DYNAMICS
4. Buffer A with 0.1% BSA: To make 50ml, combine
10ml of 5x stock buffer A, 5 0 m g BSA, and 40ml of
distilled water. Store at 4~ for 1 month.
5. Buffer A with 1% BSA: To make 10ml, combine 2 m l
of 5x stock buffer A, 100mg BSA, and 8 ml of distilled water. Store at 4~ for 1 month.
6. Secondary antibody: Colloidal gold-conjugated secondary antibody diluted 1:5 to 1:10 in buffer A
with 1% BSA.
Steps
1. After glutaraldehyde fixation (step 4 of Section
III.B.1), w a s h specimens with PBS (two brief rinses and
5 min in the third change of PBS).
2. Quench specimens by NaBH4 for 10min at room
temperature. Shake off bubbles occasionally. Rinse in
PBS (three changes, 5 min in the last change).
281
3. Remove PBS from the dish. Using cotton swabs,
wipe the buffer from the dish and coverslip, leaving
wetness on only a small (approximately 5 - 7 m m )
central area containing the locator grid. However, be
careful to avoid allowing this area to dry out. A p p l y
p r i m a r y antibody and incubate for 30-45 min at room
temperature. Rinse in PBS (three changes, 5 min in the
last change).
4. Rinse once in buffer A with 0.1% BSA. Wipe coverslips as before and apply colloidal gold-conjugated
antibody. Incubate overnight at room temperature in
a sealed dish in moist conditions. Rinse in buffer A
containing 0.1% BSA (three changes, 5 m i n in the last
change) and fix with glutaraldehyde, tannic acid,
and uranyl acetate (steps 4-6 in the Section III.B.1)
(Fig. 3).
FIGURE 3 Correlative immuno-EM of locomoting Xenopus keratocytes. The phase-contrast time-lapse
sequence was acquired with 6-s intervals. The two phase-contrast images shown (a and b) were taken I min
apart. After acquisition of the second image, cells were immediately extracted, fixed with glutaraldehyde,
quenched with NaBH4, stained with rabbit antibody to Xenopus ADF/cofilin and secondary antibody conjugated with 10nm colloidal gold, and processed for platinum replica EM. Low-magnification EM (c) shows
two keratocytes from lower left corner in b. Boxed region from c is enlarged in d. Gold particles appear as
white dots because of the reversed contrast of the original image. Distribution of gold particles demonstrates
that ADF/cofilin is excluded from the narrow zone at the extreme leading edge (Svitkina and Borisy, 1999).
282
ELECTRONMICROSCOPY
C. Critical Point Drying
The idea of the technique is to remove liquid from
the sample without exposing it to high surface tension.
This is accomplished by bringing the sample to or
above the critical point, a specific combination of
temperature and pressure for the particular liquid
where phase boundary does not exist. For most
liquids, including water, the critical point is too
extreme to be of practical use. In contrast, carbon
dioxide has a critical point at 31.3~ and 1072psi
(72.9 atm) and represents the fluid of choice for CPD of
biological samples. Because CO2 has limited solubility
in water, ethanol (or acetone) is used as a transitional
liquid, which is miscible with either water or CO2 in
any proportion.
Solutions
1. Graded ethanols: 10, 20, 40, 60, or 80% ethanol.
Combine 10, 20, 40, 60, or 80ml of 100% ethanol,
respectively, with distilled water until a final
volume of 100ml. Allow to stand until all air
bubbles are gone and temperature is equilibrated
to ambient conditions.
2. Uranyl acetate in ethanol: 0.1% uranyl acetate in 100%
ethanol. Weigh 25 mg of uranyl acetate, add 25 ml of
100% ethanol, and stir until dissolved. Use within
several hours.
3. Dried ethanol: 100% ethanol dried over molecular
sieves. Wash molecular sieves free of dust with multiple changes of water and bake overnight at 160~
After cooling, combine 50-100 g of molecular sieves
with 500ml of 100% ethanol. Seal with Parafilm.
Store at room temperature for 2 days before use.
Steps
1. If oil objectives are used for light microscopy,
remove the immersion oil from the bottom of the coverslip with cotton swabs soaked in ethanol.
2. Detach the coverslip from the bottom of a dish
and quickly transfer it into a wide petri dish filled with
water. Some silicone grease will remain on the lower
side of the coverslip. Lightly press the coverslip down
to the petri dish bottom, making sure that the grease
does not contaminate the central area of the coverslip
containing the cells of interest. Using a diamond
pencil, cut off the greased edges of the coverslip to
obtain a clean central part of the coverslip with the
locator grid. It is helpful to use a razor blade as a guide
for making cuts. Use a sharp diamond pencil and
avoid glass crumbs around the cutting area to prevent
coverslips from shattering. The optimal size of the
central piece of the coverslip containing cells of interest is 6-8 ram.
3. Place a specimen holder for CPD into a wide
beaker filled with water. Cut lens tissue into pieces
fitting the size of the holder. Put a sheet of lens tissue
on the bottom of the holder and place the coverslip
onto it. Load other coverslips one after another using
additional lens tissue sheets as spacers. The lens tissue
separates samples and helps retain a layer of liquid
over the specimens during transfer. Keep the whole
stack loose to allow for easy liquid exchange. Up to 12
coverslips with dimensions 6-8 m m may be processed
simultaneously. Overloading the holder makes the
exchange of liquid difficult. Loosely put on a lid to
prevent the last sheet of lens tissue from flowing away.
4. Put a stirrer bar into a 50-ml beaker. Place a wire
scaffold over the stirrer. Add 10% ethanol in amount
sufficient to cover the specimen holder when it is
placed onto the scaffold. Quickly transfer the holder
from water to the beaker (Fig. 1). Stir for 5 min.
5. Prepare another beaker with 20% ethanol in the
same way. Transfer the holder and stir for 5min.
Repeat this step for 40, 60, 80, and twice for 100%
ethanol. Two sets of beaker/stirrer bar/scaffold are
sufficient for dehydration, as they can be alternated in
successive steps.
6. Place holder into uranyl acetate in ethanol and
incubate for 20min. No stirring is necessary.
7. Prepare beaker as in step 4 but with 100%
ethanol, put in holder, and stir for 5 min. Repeat once
more. Then, repeat twice with dried 100% ethanol.
8. Fill the specimen chamber of the CPD device
with dried 100% ethanol. The amount of ethanol in
CPD chamber should be just enough to cover the
holder. Place holder into the chamber. If the CPD
device is equipped with a stirrer, put a stirrer bar
underneath the holder. Close chamber and open CO2
cylinder and inlet valve on CPD machine. Cool down
the chamber to 10-15~ to keep CO2 in liquid state.
Maintain this temperature until the heating step. Turn
stirrer on. Wait until the chamber is filled.
9. Slightly open exhaust valve for 30 s, keeping inlet
valve open to allow for exchange of ethanol to liquid
CO2. If the CPD is not equipped with a stirrer, shake
CPD manually during this step. Close exhaust valve.
Repeat this washing step 10 times every 5min to
remove all traces of ethanol. Keep the level of CO2
always above the upper edge of the holder.
10. Turn off stirrer and cooler. Turn on heat to raise
pressure and temperature above the critical point for
CO2, usually until 40~ and 1200psi (80atm). Then
slowly release pressure by opening exhaust valve. A
fast decrease of pressure may cause condensation of
CO2 back to liquid and ruin the dried samples.
11. Remove holder from the CPD chamber and
immediately place it in a sealed desiccated container.
CORRELATIVE LIGHT AND EM STUDIES OF CYTOSKELETAL DYNAMICS
Dried cells can easily absorb moisture from air,
which will introduce artifacts similar to those created
by air drying. Therefore, it is important to keep
samples inside the desiccator until ready for replica
preparation.
D. Platinum Replica Preparation
1. Shadowing
Rotary shadowing at an angle creates a gradation of
metal thickness depending on the 3D organization of
the sample. Platinum is a popular metal for vacuum
evaporation because it represents a reasonable compromise between melting temperature and grain size.
Platinum grains deposited onto the specimen surface
are not cohesive and can be distorted easily during
subsequent manipulations or under the electron beam.
The platinum layer should be stabilized by carbon,
which forms a cohesive film and thus keeps platinum
grains in place. The specific procedures for platinum
and carbon shadowing depend on the particular
device. Therefore, we describe just some important
issues.
Mounting of Coverslips
Rotation of the stage will dislodge samples if they
are not secured on the stage. Double-sided sticky tape
is too strong and does not permit easy and safe detachment of samples, especially after being in vacuum. To
make a mild mounting tape, sandwich double-sided
tape between sticky parts of two Post-It notes so that
the glued side of paper sheets is exposed. Cut off the
unglued paper. To mount coverslips, attach a piece
of this sandwich to the evaporator stage and attach
coverslips. It is sufficient to attach just a corner or an
edge of a coverslip to the paper. It is helpful to put
marks on the paper to identify samples.
Platinum Shadowing
Source. Our system is set up to use platinum wire
wrapped around tungsten wire as a source for shadowing. When voltage is applied, the tungsten wire
heats up and the platinum melts and evaporates. Alternatively, platinum-carbon pellets can be used as a
source. An advantage of pellets is that they produce
finer grains.
Angle. Low angles from the source of platinum to
the specimen stage provide high contrast, but reveal
only the very top of the sample. High angles result in
less contrast but allow for better visualization of the
cell interior because of increased penetration of metal
into deep hollows. We found a 45 ~ angle to be most
283
useful for whole mount cytoskeleton preparations, as
it represents a reasonable compromise between contrast and penetration.
Thickness. Thicker coating reduces resolution but
increases contrast and 3D range. In our experiments, a
platinum layer thickness of 2.5-2.8 nm produces a fair
balance between contrast and resolution. Thickness of
the platinum layer can be monitored using a quartz
crystal-based thickness monitor. If a thickness monitor
is not available, approximate settings of the system
may be established by a trial-and-error approach. In
our system, 10mg of platinum wire completely evaporated from a distance of 100mm produced a layer of
the required thickness.
Carbon Coating
Evaporate carbon at 90 ~ with or without rotation to
obtain a 2- to 3-nm-thick layer. The thickness of carbon
is not very critical, as it is practically transparent
to electrons. However, a layer thicker than 10nm
becomes visible and interferes with the formation of
image. Too thin a carbon layer may be insufficient for
stabilization and result in crumbling of replicas after
the removal of coverslips.
2. Mounting of Replicas on Grids
Platinum-carbon replicas of the cytoskeleton are
released from the coverslip with hydrofluoric acid. If
cell areas that are going to be studied are thin and have
low electron density, such as lamella in spread cultured cells (Fig. 4), removal of glass is sufficient. For
thick and electron-dense cell regions, organic components can be depleted with a strong oxidative agent,
e.g., household bleach. For correlative microscopy, it is
easier to select the area of interest while the replica is
still attached to the coverslip. After drying and metal
coating, cells have good contrast and are visible even
under the dissection microscope.
Solutions
1. Hydrofluoric acid: ~5% HF in water. Concentrated
(49%) HF solution is supplied by the manufacturer in
a plastic dispenser bottle. Work with HF in a fume
hood, use plastic (not glass) dishes and pipettes, and
wear gloves. Store in the fume hood. Prepare working
solution in a 12-well dish (diameter 25 mm) before use.
Drip several drops (-0.5-1.0ml) of concentrated HF
from the dispenser bottle into a well. Add distilled
water almost to the top.
2. 0.01% Triton X-IO0: Take 10ml of distilled water
and add 10~tl of 10% Triton X-100. Store at room temperature not longer than 1 month.
284
ELECTRONMICROSCOPY
FIGURE 4 Correlative fluorescence and EM of mouse melanoma B16F1 cells. The cell shown was transiently transfected with EGFP-fascin (a) and, after extraction and fixation, stained with Texas red phalloidin
(b). After EM processing, the same cell was identified at low magnification (c). Lighter background at the
upper left corner is due to gold evaporation through a hole of the locator grid. (d) High-magnification view
shows actin filament organization in the leading lamellipodium. Several microspikes within lamellipodium,
which are enriched in fascin (see a), have actin filaments organized into tight bundles, whereas lamellipodium
between microspikes, which is depleted in fascin, has actin filaments organized into the dendritic network.
3. Clorox bleach (optional): Dilute in distilled water
1:2 to 1:10 d e p e n d i n g on the strength of bleach.
Steps
1. Using mild double-sided tape (see earlier discussion), immobilize a platinum-carbon-coated coverslip on the bottom of a wide petri dish with cell side
up, leaving a region of interest unobstructed.
2. Under the dissection microscope, localize cells of
interests using the gold pattern. Make cuts with any
sharp tool (razor blade or needle) in the platinumcarbon layer a r o u n d cells of interest. Continue the cuts
up to the edges of the coverslip to facilitate release of
the selected area from the rest of the replica.
3. Float a coverslip with cell side up onto the
surface of the HF in a well. In minutes the coverslip
falls down, leaving the replica floating. After separation of the coverslip, the replica falls apart along the
introduced cuts. The pattern of the gold s h a d o w i n g on
the resulting pieces helps identify the desired replica
fragments.
4. Fill another well with distilled water (~5ml).
A d d ~2gl of 0.01% Triton X-100. Using a platinum
loop, transfer replica pieces onto the surface of water.
Traces of detergent in the water prevent the replica
from breaking apart, which usually h a p p e n s because
of a large difference in surface tension between HF and
water. An overdose of detergent, however, can result
in shrinkage and d r o w n i n g of replicas. Wait I min or
more.
5. Fill a well with distilled water. Transfer replica
pieces onto the surface of pure distilled water. Wait
l min or more. For electron-dense specimens, go
through additional steps:
a. Fill a well with diluted household bleach and
transfer replica pieces onto its surface. Wait 2
CORRELATIVELIGHTAND EMSTUDIESOF CYTOSKELETALDYNAMICS
to 20min d e p e n d i n g on the cell type and the
strength of the bleach.
b. Fill another well with distilled water. Transfer
replica pieces onto the surface of pure distilled
water. Wait l min or more. Repeat the step
once more.
Note: Depletion of organic material by bleach is not
compatible with i m m u n o g o l d labelling, as it causes
the degradation of antibody associated with colloidal
gold particles and consequent elimination of gold label
from replicas.
6. M o u n t replica pieces onto Formvar-coated EM
grids with lower side of the replica to the Formar film.
Use low m e s h or single slot grids to reduce the chance
of getting the region of interest onto a grid bar. Control
u n d e r the dissection microscope is helpful for the targeted m o u n t i n g of replicas on grids.
7. Examine samples in TEM. (Fig. 4) Present images
in inverse contrast (as negatives) because it gives a
more natural view of the structure, as if illuminated
with scattered light.
285
tension (Ris, 1985). To avoid this problem, always keep
a layer of liquid over the specimens w h e n they are
transferred from one solution to another. The problem
m a y also occur because of incomplete replacement of
water to ethanol or ethanol to liquid CO2 during dehydration and CPD. A n y remaining traces of ethanol or
water d r y out below their critical points after CPD and
ruin the structure. Another possible reason is high
ambient humidity. Dried samples are highly hygroscopic. They m a y absorb moisture from air, which
will subsequently dry below the critical point. Loading
the evaporator is a step w h e n samples are most susceptible to humidification, as it takes some time to get
coverslips from the CPD holder and m o u n t them onto
the evaporator stage. Keep h u m i d i t y in the room as
low as possible. In our experience, it is sufficient
to keep it u n d e r 50%. Also, prepare evaporator for
coating in advance, before getting samples from the
desiccator.
References
IV. PITFALLS
1. Cytoskeletal elements look distorted. Most likely,
extraction was not performed gently enough. Explore
different extraction conditions comparing living and
extracted cell images.
2. Cytoskeletal elements look fragmented. One possible
reason is inadequate fixation. Check your reagents for
fixation capacity. Another possibility is p h o t o d a m a g e
or phototoxicity during live cell imaging. Decrease
light a n d / o r exposure and close field d i a p h r a g m as
m u c h as possible to the area of interest during
sequence acquisition.
3. Cytoskeletal elements look flattened and fused with
each other. This is an artifact introduced by surface
Lindroth, M., Bell, P. B., Jr., et al., (1992). Preservation and visualization of molecular structure in detergent-extracted whole mounts
of cultured cells. Microsc. Res. Tech. 22, 130-150.
Ris, H. (1985). The cytoplasmic filament system in critical pointdried whole mounts and plastic-embedded sections. J. Cell Biol.
100, 1474-1487.
Svitkina, T. M., and Borisy, G. G. (1998). Correlative light and electron microscopy of the cytoskeleton of cultured cells. Methods
Enzymol. 298, 570-592.
Svitkina, T. M., and Borisy, G. G. (1999). Arp2/3 complex and actin
depolymerizing factor/cofilin in dendritic organization and
treadmilling of actin filament array in lamellipodia. J. Cell Biol.
145, 1009-1026.
Svitkina, T. M., Verkhovsky,A. B., et al., (1995). Improved procedures
for electron microscopic visualization of the cytoskeleton of cultured cells. J. Struct. Biol. 115, 290-303.
Svitkina, T. M., Verkhovsky,A. B., et al., (1997). Analysis of the actinmyosin II system in fish epidermal keratocytes: Mechanism of
cell body translocation. J. Cell Biol. 139, 397-415.