Glycerol Spraying/Low-Angle Rotary
Heavy-metal replication (Williams and Wyckoff,
1945) is one of the oldest and yet most effective
methods to provide enhanced contrast when imaging
biological specimens in a transmission electron microscope
(TEM) (for review, see Fowler and Aebi, 1983;
Mould et al.
, 1985; Nermut and Eason, 1989). This technique
requires efficient adsorption of the sample to a
specimen support so that the biomolecules or particles
to be visualized become well dispersed and distributed
evenly. Moreover, to minimize shadow-casting
artifacts produced by corrugations of the specimen
support, it should ideally be smooth at atomic dimensions.
Mica fulfills both these requirements and hence
emerges as an ideal specimen support. For a good
replica, the contrast-enhancing metal should produce
a fine grain, detach easily from the specimen support,
and be stable in the electron beam. An example of a
metal that favorably meets these three criteria is
The combination of mica and platinum was first proposed
by Hall (1956) in his 'mica replication' technique.
Hall sprayed different solutions of biomolecules (i.e.,
an adenosine phosphate polymer, collagen, DNA, and
fibrinogen) onto a piece of freshly cleaved mica. The
mica was then dried in a vacuum evaporator and subsequently
metal casted at a low angle (i.e., 5-15° with
platinum. The metal layer on the mica was stabilized
by backing it with a layer of each of SiO and collodion.
The resulting replica was then floated off the mica onto
distilled water, picked up on a specimen support grid,
and imaged in a TEM (Hall, 1956). More disperse
spreading and even distribution of the biomolecules on
the mica surface were achieved by adding glycerol to
the sample before spraying it (Tyler and Branton, 1980;
Fowler and Erickson, 1983). For their modification of
Hall's technique, Tyler and Branton coined the term glycerol drying
. Specifically, they added glycerol to the
sample before spraying it onto the mica and backed the
metal replica with carbon only. The more accurate and
appropriate term used now is glycerol sprayingflowangle
rotary metal shadowing
Glycerol spraying/low-angle rotary shadowing has
been claimed to preserve native conformations comparably
to cryotechniques (Mould et al.
, 1985; Nermut
and Eason, 1989). It has been used time and again to
characterize complex macromolecules at near-native
conditions and especially also proteoglycans (Moergeli et al.
, 1985; Rodriguez et al.
, 1995; Scheel et al.
Hofmann et al.
, 2000; Griffith et al.
, 2002). Indeed,
a stabilization of the native confirmation by glycerol
against temperature-, pH-, or organic solvent-induced
changes was corroborated by electron spray mass
spectrometry (Grandori et at., 2001).
For glycerol spraying, droplets of a glycerolcontaining
solution are vaporized using air pressure
and sprayed against a mica surface. The droplets spread
on impact and retract rapidly due to the high surface
tension of the glycerol-containing solution. Molecules
or particles adsorbed to the mica surface are left
behind with little or no solvent surrounding them.
Therefore, they dry almost instantaneously. The dried,
retracted sample droplets produce the characteristic
"droplet centers" (see Fig. 3a) that are observed with
glycerol-sprayed preparations at low magnification.
These droplet centers represent a residue that is
formed by the salts in the sample buffer and by unabsorbed
protein and other debris that is swept off the
mica by the advancing or retracting drop (Tyler and
Specimens that are suitable for glycerol spraying/
low-angle rotary metal shadowing range from
single protein (e.g., myosin) and DNA molecules
to relatively stable biopolymers or supramolecular
assemblies such as intermediate filaments or
protein/glycosaminoglycan complexes. The achievable
resolution is a few nanometers and thus sufficient
to reveal the overall size and shape and/or the principal
domain architecture of many biomolecules.
II. MATERIALS AND
|FIGURE 1 Standard equipment for glycerol spraying/low-
metal shadowing. Two adjustable pipettes:
(a) Gilson Pipetman P20,
and (b) Gilson Microman M25,
together with their respective tips;
(c) a pair of scissors;
(d) copper specimen grids; (e) spot plate; (f)
(g) 25-µl glass micropipettes with pipettor; (h) Pasteur
pipettes; (i) Eppendorf tubes; and a pair each of (j) fine,
forceps, and of (k) fine, bent forceps.
For routine applications of glycerol spraying/lowangle
rotary metal shadowing, the following items,
gadgets, and equipment are required.
- Gilson Pipetman adjustable pipette P20, with tips
(Fig. 1a), and Gilson Microman adjustable pipette M25,
with tips (Fig. 1b) (http://www.gilson.com/)
- Vortex apparatus
- Pair of scissors (Fig. 1c).
- Double-sided adhesive Scotch tape
- Specimen support grids (Fig. 1d): For example,
from Pelco International (http://www.pelcoint.com/)
(typically, 400 mesh/in, copper grids, 3.05mm in
diameter and 0.7mm thick)
- Spot plate (Fig. 1e): For example, from BAL-TEC
(http://www.bal-tec.com/) (Cat. No. B 8010 030 83)
- Mica sheets (Fig. 1f): For example, from BALTEC
(Cat. No. BU 006 027-T)
- 25-µl glass micropipettes with pipetter (Fig. 1 g)
- Pasteur pipettes (Fig. 1h)
- Eppendorf tubes (Fig. 1i)
- Precision forceps: For example from Electron
Microscopy Sciences (http://www.emsdiasum.com/
ems/). A pair of fine, straight forceps (Fig. 1j) and a
pair of fine, bent forceps (Fig. 1k).
- Compressed air at a pressure of 0.8 bar
- Custom-built spray apparatus with air-pressure
controller (Figs. 2d and 2e). The major elements or
pieces to be manufactured or purchased include
A polyvinyl chloride (PVC) frame mounted onto an
A Pasteur pipette connected to a compressed air
supply via an air pressure/volume controller
unit so that a set volume of a fine stream of air is
produced at its nozzle by the push of the release
button on the controller
A clamp mounted on a post to fasten a 25-µl glass
micropipette containing the sample so that it
points to the center of the stream of pressurized
A metal sphere on a height-adjustable post so that
it can be placed into the center of the fine air
stream and disperse the sample drops evenly
A curved glass tube that traps the large sample
droplets, aggregates, and other heavy particles
A square piece of freshly cleaved mica that is fastened
with a piece of double-sided adhesive tape
below the curved glass tube
- Glow-discharge apparatus: For example,
custom built as detailed by Aebi and Pollard (1987)
(see also article by Baschong and Aebi) or SCD 005
(Cat. No. BU GO5 750 116-T) from BAL-TEC.
- High-vacuum evaporation unit: For example,
BAE 080-T (Cat. No. BU P03 527) from BAL-TEC
- High-vacuum pumping unit with rotary
- Vacuum measuring device for rough and
- Evaporation unit with controller, high current
supply, and cross-shaped vacuum chamber
- Accessories for BAL-TEC high-vacuum evaporation
- Cold trap for BAE 080-T (Cat. No. BB 176 942-
- Quick-release flange WF 206, including EK-
030 electron gun (Cat. No. BU 007 038-T)
- Quick-release flange WF 211, rotary table
(Cat. No. BB 192 281-T)
- Quick-release flange WF 204, C-evaporator
(Cat. No. BB 192 276-T)
- Digital quartz-crystal thickness meter QSG
060 (Cat. No. BU M01 259)
- Electron beam evaporation device EVM 030,
(Cat. No. BU S04 000)
- Materials for BAL-TEC high-vacuum evaporation
- Carbon rods for Pt/C evaporator (Cat. No.
BU 006 101-T)
- Platinum inserts (Cat. No. BU 006 103-T)
- Carbon rods for C-evaporator (Cat. No. BU
- Tungsten cathodes (Cat. No. BU 020 023-T)
- Liquid nitrogen
- Glycerol 100% anhydrous: For example, from Fluka
(http://www.sigmaaldrich.com/)(Cat. No. 49770)
- Sodium hypochlorite solution, 13-15%, technical
grade: For example, from Siegfried
siegfried.ch/) (Cat. No. 180550-01)
- Hold a piece of mica with a pair of straight forceps
and cut it into square pieces (5-7mm side length)
with a pair of scissors (Fig. 2a).
- Dilute the sample to a concentration of about 10-
100nM (0.1-1.0 mg/ml for most proteins) and pipette
20µl into an Eppendorf tube.
- Add 8.6µl of 100% glycerol to the sample (i.e., 30%
final glycerol concentration) using the Gilson Microman
M25. Mix thoroughly with the pipette and
- Draw 10 µl of the glycerol-containing solution into a
25-µl glass micropipette and mount it on the spray
apparatus (Figs. 2d and 2e).
- With two pairs of forceps, cleave the mica into two
sheets (Figs. 2b and 2c).
- With its freshly cleaved side up, place the mica
below the bent glass tube of the spray apparatus.
During spraying, the mica sheet should either be
fixed with a piece of double-sided adhesive tape as
shown in Fig. 2e or held in position with a pair of
- Briefly (i.e., for about 1s) press the button of the airpressure
control unit (depicted in Fig. 2d) to spray
the sample suspension onto the freshly cleaved mica.
Note: The metal sphere placed in the spray path disperses
the sample droplets evenly. The curved glass
tube traps remaining large droplets, aggregates, and
other heavy particles that will not follow the air flow
when the spray path changes its direction by 90° .
- Repeat spraying in the same way with another 10 µl
of suspension onto the second piece of mica, etc.
|FIGURE 2 Selected steps of the glycerol spraying/low-angle rotary metal shadowing technique. (a) A piece of mica
held with a pair of straight forceps is cut into square pieces with a pair of scissors. (b and c) Using forceps,
the mica is next cleaved into two sheets. (d and e) The glycerol-containing solution is drawn into a 25-µl glass
micropipette that is mounted on the spray apparatus such that it points to the center of the stream of pressurized
air that is focused by a Pasteur pipette. (f) The Pt/C-shadowed and C-backed mica is slowly
immersed into water, and (g) the mica sheet is removed once the Pt/C+C-replica is floated off. (h) The replica
is then placed on a specimen grid with a pair of forceps.
B. Metal Evaporation
C. Floating off the PT/C+C Replica
- Switch on the thin film quartz monitor.
- Set up the electron gun for Pt/C evaporation and
the carbon evaporation unit for C-evaporation
according to the instruction manual of the supplier.
- The recommended working distance between the
Pt/C tip in the electron gun and the middle of the
table is 12 cm and the tilt angle of the table relative
to the evaporation axis should be around 5° . For
the C tips in the C evaporation unit, the recommended
working distance is the same but the tilt
angle should be 80-90° .
- Mount the mica with double-sided adhesive tape
onto the table. Make sure that the sprayed side of
the mica is up!
- Mount the table onto the rotating base in the
vacuum chamber and start evacuating.
- After 30 min, the vacuum meter should read better
than 2 × 10-5 mbar.
- Pour liquid nitrogen into the Meissner trap.
- After an additional 5min, the vacuum meter
should read better than 6 × 10-6 mbar.
- Degas Pt/C tip according to the instruction
manual of the supplier.
- Start the motor of the rotating base (set to about
- Set the thin film quartz monitor to zero.
- Start evaporating Pt/C according to the instruction
manual. Open the manual shutter.
- Read the frequency on the thin film quartz
- After the frequency has changed by 300 Hz, close
the manual shutter and stop evaporation.
- Evaporate C according to the instruction manual
of the supplier.
- On a white reference the carbon layer should look
deep brown (cf. Figs. 2f and 2g).
Note: It is advisable to calibrate current and evaporation
times of the power supply so that standard settings
can be used.
- Turn the rotating table off, break the vacuum, and
remove the rotating table from the vacuum chamber.
D. Electron Microscopy
- Fill the wells of the spot plate (Fig. le) with distilled
water (Fig. 2f).
- As illustrated in Fig. 2f, slowly immerse the mica at
an angle of ~45° holding it firmly with the forceps.
- Submerge the mica completely and remove it when
the Pt/C+C-replica is floating on the water surface
- With a pair of forceps, submerge a freshly glowdischarged
specimen grid, place it underneath the
Pt/C+C-replica, and use it as a sieve to collect the
replica piece by piece.
- Dry the specimen grids by layering them with the
film side up onto a piece of filter paper.
- The specimen grids are now ready for inspection in
|FIGURE 3 Electron microscopy of glycerol-sprayedflow-
angle rotary metal-shadowed preparations. (a) At low
magnification, very prominent features of glycerol-
sprayed/low-angle rotary metal-shadowed preparations
are the "droplet centers" formed by the salts in the buffer,
unabsorbed protein, and debris that are swept off
mica by the advancing and/or retracting drop of the
specimen/glycerol solution. (b) When moving from
center of such a droplet center outward, a transition from
a coarse and irregular to a smooth and clean
is observed. Well-spread molecules are found in the zone
that shows a clean background (demarcated
lines). (Inset) A higher magnification view that reveals
head-to-tail polymers of myosin-like
Heitlinger et al., 1991).
- Identify droplet centers as shown in Fig. 3a at a
low magnification of, e.g., 5000×. Note: The droplet centers are easy to find if your sample is kept in nonvolatile
buffers and/or contains salts. The droplet
center size varies from 5 to 50 µm in diameter. Choose
droplet centers with a size of about 5-10µm.
- Increase the magnification to about
20,000-50,000×. Set coarse focusing and then slowly
move from the center toward the edge of the
- Dashed lines in Fig. 3b highlight the sharp transition
from salt crystals to a clean background that
is typically observed over increments of the droplet
center distance by 0.5-2 µm. In this "clean belt" around
the droplet center well-spread and well-preserved
protein particles are usually found readily (Fig. 3b,
- Replica resists detaching from the mica. Occasionally,
the Pt/C+C replica sticks to the mica. The
replica usually detaches more easily after the mica has
been incubated in a moist atmosphere at 37°C for
30min. Alternatively, the replica may also be floated
off onto 6% sodium hypochlorite rather than water;
however, after this treatment, the replica has to be
transferred with a platinum loop to distilled water and
allowed to incubate for 10 min. One reason for frequent
sticking of the Pt/C+C replica to the mica may be too
good a vacuum during C evaporation. Breaking the
vacuum after Pt/C evaporation, reevacuation to 7 × 10-5 mbar, and subsequent C evaporation may solve
- Preparation not satisfactory: (a) Specimen
dependent: The specimen may not be optimal for glycerol
spraying. Some specimens, including actin filaments,
are relatively sensitive to shearing forces and
may thus break into small pieces on spraying. (b) Buffer
dependent: Many buffers and solutions can be used but
detergents or high concentrations (i.e., ≥2M) of urea
or guanidine hydrochloride cause problems through
eutectic effects. In this case, the buffer should be
changed or diluted.
- Phase separation after addition of glycerol. For
good results, it is essential that the sample is a solution
that contains glycerol. In case of phase separation, different
buffers should be tried. If volatile buffers (e.g.,
ammonium acetate) are used, the evaporating unit
should be evacuated for 2-3h rather than for 30min
before metal evaporation (see earlier discussion).
This work was supported by the M. E. Mueller
Foundation of Switzerland and the Canton
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