Self-pressurized rapid freezing (SPRF): a novel

Journal of Microscopy, Vol. 235, Pt 1 2009, pp. 25–35
Received 17 December 2008; accepted 31 March 2009
Self-pressurized rapid freezing (SPRF): a novel cryofixation method
for specimen preparation in electron microscopy
J.L.M. LEUNISSEN∗ & H. YI†
∗ AURION ImmunoGold Reagents, Wageningen, The Netherlands
†Emory School of Medicine Electron Microscopy Core, Emory University, Atlanta, Georgia 30322,
U.S.A.
Key words. Cryo fixation, EM preparation, HPF, SPRF.
Summary
A method is described for the cryofixation of
biological specimens for ultrastructural analysis and
immunocytochemical detection studies. The method employs
plunge freezing of specimens in a sealed capillary tube
into a cryogen such as liquid propane or liquid nitrogen.
Using this method a number of single-cell test specimens
were well preserved. Also multicellular organisms, such as
Caenorhabditis elegans, could be frozen adequately in low ionic
strength media or even in water. The preservation of these
unprotected specimens is comparable to that achieved with
high-pressure freezing in the presence of cryoprotectant. The
results are explained by the fact that cooling of water in a
confined space below the melting point gives rise to pressure
build-up, which may originate from the conversion of a
fraction of the water content into low-density hexagonal ice
and/or expansion of water during supercooling. Calculations
indicate the pressure may be similar in magnitude to that
applied in high-pressure freezing. Because the specimens are
plunge cooled, suitable cryogens are not limited to liquid
nitrogen. It is shown that a range of cryogens and cryogen
temperatures can be used successfully. Because the pressure
is generated inside the specimen holders as a result of the
cooling rather than applied from an external source as in
high-pressure freezing, the technique has been referred to as
self-pressurized rapid freezing.
Introduction
Physical fixation of biological specimens is the basic principle
underlying a range of specimen preparation methods in
electron microscopy aimed at preserving ultrastructure,
molecular functionality and recognition, as well as electrolyte
distribution. Several methods have been developed involving
Correspondence to: Jan L.M. Leunissen. E-mail: [email protected]
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Journal compilation freezing in the presence of a cryoprotectant, with high heat
extraction rates, and under hyperbaric pressure.
For
ultrastructural
and
immunocytochemical
investigations, by far the largest share of all cryo-based
specimen preparations, adequate cell and tissue cryofixation
may be achieved by applying cryoprotectants (e.g. Tokuyasu,
1973; Franks, 1977; Gilkey & Staehelin, 1986). High cooling
rates (>105◦ Cs−1 ) allow vitrification of thin layers only
(Dubochet & McDowall, 1981), which is not always sufficient
for general ultrastructural and immunocytochemical
detection studies. High-pressure freezing (HPF) as presented
by Moor & Riehle (1968) is a technique that allows for
a depth of 100–300 μm from the specimen surface to be
frozen without detectable ice crystallization damage (Dahl &
Staehelin, 1989; Studer et al., 1989; Vanhecke et al., 2008).
At 200–210 MPa pressure high cooling rates are no longer a
prime requirement. All currently available HPF instruments
use liquid nitrogen as the cryogen.
HPF has become an accepted technique for the structural
preservation of pro- and eukaryotic cells, multicellular
organisms and tissues, allowing further processing with
cryosubstitution (Monaghan et al., 1998; Hawes et al.,
2007; Buser & Walther, 2008; Triffo et al., 2008), freeze
fracturing (Dahl & Staehelin, 1989; Walther, 2003) and
cryoultramicrotomy (van Donselaar et al., 2007). Despite
its suitability to freeze unprotected specimens, in many
published applications HPF is combined with the use of a nonpenetrating cryoprotectant such as dextran or serum albumin
to further support cryopreservation (Dubochet, 1995). In
the absence of cryopreservatives crystalline ice is formed in
the extracellular space, which affects preservation (Erk et al.,
1998; Dubochet, 1995).
HPF is based on the principle of Le Chatelier and
Braun which postulates that if a system at thermodynamic
equilibrium experiences a change in one of the physical
parameters involved, then the equilibrium will shift in order
to minimize that change. In HPF this principle explains how
an externally applied pressure of between 200 and 210 MPa
26
J.L.M. LEUNISSEN AND H. YI
prevents water from expanding into low-density ice upon
cooling.
The present manuscript introduces the self-pressurized
rapid freezing (SPRF) method based on the same principle
of Le Chatelier and Braun. It uses the tendency for water
inside the specimen container to expand upon cooling, thereby
generating pressure intrinsically instead of using an external
hydraulic system. This pressure is likely to be the result of
two processes: ice crystallization and/or supercooling which
is the phenomenon that water remains liquid at temperatures
well below the melting point of ice. The ambient pressure
polymorphs of ice as well as supercooled water have a lower
density than water at 0◦ C. Therefore, upon cooling a specimen
that is enclosed in a confined space below 0◦ C pressure will be
generated. In practice, pressures comparable to those applied
in HPF can be generated in this way (Hayashi et al., 2002).
In this manuscript, we report how this phenomenon can be
exploited for ultrastructural preservation studies.
ensuring a tight fit. The micropipette volume was set to a
few microlitres in excess of the tube capacity (1.54 μL) to
allow for slight overfilling of the capillary (4–5 μL). The open
end of the capillary was inserted into the specimen suspension
and the suspension was drawn into the tube slowly using
the micropipette action, preventing air bubbles from forming
inside the capillary. If necessary the process was repeated.
Capillary tube sealing
The filled capillary tube, while mounted in the micropipette tip,
was withdrawn from the suspension. The open end was sealed
by clamping a length of about 1–1.5 mm shut using needlenose pliers with flat jaws. Next, while pressing the pipette
piston down, the capillary was removed from the micropipette
tip and the open capillary end sealed by tightly clamping a
length of about 1–1.5 mm shut using the pliers. For controls,
tubes were filled as described but not sealed.
Cryoprocessing
Materials and methods
Specimens
Caenorhabditis elegans (C. elegans) nematodes were a kind gift of
Dr. Guy Benian, Dept. of Pathology and Laboratory Medicine,
Emory University, Atlanta. Bacterial cultures (Anoxybacillus
flavithermus and Geobacillus stearothermophilus) were kindly
provided by Hannah Heinrich, MSc (Dept. of Biochemistry,
University of Otago). Yeast cells (Saccharomyces cerevisiae)
were kindly provided by Dr. Paul Doetsch and Dr. Natalya
Degtyareva, Dept. of Biochemistry, Emory University, Atlanta.
Bacteria, yeast cells and nematodes were collected and frozen
in distilled water. Alternatively yeast cells were collected and
frozen in 10 mM phosphate buffer, 145 mM NaCl, pH 7.4 or in
0.3 M sucrose solution in water.
Specimen holders
Capillary copper tubes 16 mm in length and with an outer
diameter (o.d.) of 0.65 mm and an inner diameter (i.d.) of
0.35 mm were obtained from Leica Austria (Leica part number
16706871) (Leica, Vienna, Austria). These capillary tubes
contain 1.54 μL when they are fully filled with specimen.
In addition copper capillaries with nominal dimensions of
0.8 mm o.d., 0.3 mm i.d. and 1.3 mm o.d., 0.4 mm i.d.
(Albion Alloys Ltd, Bournemouth, UK) were used. Capillary
tubes were pre-cleaned by sonication in 0.1% Triton-X-100 to
hydrophilize the interior surface to prevent air bubbles from
sticking and rinsed in distilled water before use.
Capillary tube mounting and specimen loading
Capillary tubes were inserted into a disposable pipette tip
mounted on a 0–20 μL micropipette. Part of the pipette tip
was cut off to facilitate mounting of the capillaries while
Sealed capillaries containing specimens were either dipped
manually or plunged into liquid nitrogen or liquid propane
(−180◦ C or −120◦ C) or melting isopentane using either an
EMS Plunge 002 (EMS, Hatfield PA, USA), a Leica/Reichert
KF-80 or a Leica CPC (both Leica, Austria) as follows.
Capillaries, held with fine forceps, were inserted into the
cryogen horizontally to assure that the clamped ends were
inserted simultaneously. Specimens were left in cryogen for a
minimum of 15 s after which they were stored under liquid
nitrogen awaiting further processing.
Further processing and freeze substitution
Capillaries were cut under liquid nitrogen into approximately
1–2-mm-long segments with a pre-cooled capillary tube cutter
(DT-001, Wujiang Dunnex Tools Co. Ltd, Suzhou, China).
Using a capillary tube cutter prevents the capillary from
collapsing during cutting. Alternatively they were cut into
6-mm-long segments and sliced open under liquid nitrogen
to expose the contents using the Leica HPF cryotools (Leica
product package 16706855). Capillary segments containing
specimen were then transferred under liquid nitrogen into a
cryo vial that contained frozen substitution medium. Samples
were freeze-substituted (van Harreveld & Crowell, 1964) in a
Leica AFS automatic freeze substitution apparatus. Segments
cut from two capillary tubes were sometimes pooled to ensure
sufficient specimen volume for further processing.
Substitution took place in acetone, containing 2% OsO 4 and
0.1% uranyl acetate. Typically, specimens were kept at −90◦ C
for 72 h, occasionally turning the substitution vials 360◦ to
make sure dissolved ice did not accumulate at the bottom
of the vials containing the substitution solution. Hereafter
the temperature was raised at a rate of 2◦ C/hr to −60◦ C,
−30◦ C and 0◦ C and kept at each level for 8 h in the original
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Journal compilation SELF-PRESSURIZED RAPID FREEZING (SPRF)
substitution medium. Vials containing specimen were then
removed from the AFS and left at 4◦ C for 1 h before being
brought to room temperature for subsequent processing.
Substitution medium containing specimen material was
transferred to an Eppendorf tube and centrifuged. Part of the
substitution medium was removed with care to avoid specimen
material at the tip of the tube from being disturbed. Acetone
(100%) was added to the capillary segments remaining in the
original cryo vial. Any sample material remaining inside the
capillary segments was pushed out using an eye lash probe or
flushed out with a micropipette under a dissecting microscope.
Capillary segments were then removed and all specimen
material in acetone was collected into the corresponding
Eppendorf tube for centrifugation.
Specimens were rinsed at room temperature three times
with 100% acetone and then embedded in epoxy resin
(Quetol or Spurr), ultrathin sections were post-contrasted
using aqueous uranyl acetate for 10–15 min and lead citrate
for 8–10 min at room temperature.
Results
The effect of freezing on capillary tubes
The success of SPRF depends on generating pressure inside
the clamp-sealed specimen capillary tube and holding that
pressure for the time it takes to cryopreserve the specimen.
Tests were therefore carried out to verify that the sealed
ends of the tubes (Fig. 1) would not leak and that
there was no deformation of the tubes during the cooling
process. Cooling by dipping or plunging in liquid nitrogen
or propane, where the clamp-sealed capillary is inserted
parallel to the cryogen surface, resulted in no noticeable
deformation or bursting of the capillaries. The outer diameter
of both empty and distilled water-filled capillaries before
and after freezing as observed in cryo-SEM at a specimen
stage temperature of −180◦ C is not noticeably different
27
from the values at room temperature. Cryo-SEM Scanning
Electron Microscopy examination revealed no leakage from
the clamped ends of water-filled frozen capillaries (Fig. 1). At
high magnifications it appears that the slit width is about
1 μm or less. No signs of leakage were apparent as a result of
pressure developed inside the capillaries. By contrast, dipping
the tubes perpendicular to the cryogen surface often resulted
in leaking of the top seal.
Open capillaries clearly showed expansion of the liquid from
inside the tube upon cooling such specimens in cryogen.
Ultrastructural preservation
Freeze-substituted bacteria, yeast cells as well as C. elegans
nematodes were observed after freezing in clamp-sealed
capillary copper tubes using either liquid nitrogen at −196◦ C
or liquid propane at −180◦ C or −120◦ C (Figs 2–5).
Thermophilic bacteria Anoxybacillus flavithermus and
Geobacillus stearothermophilus showed typical ultrastructure of
gram-positive bacteria with clearly defined plasma membrane,
peptidoglycan cell wall and polysaccharide capsule. Within
these bacteria the cytoplasm appeared with no apparent
segregation. The most distinct structure inside the bacteria
was ribosomes. On occasion the nucleoid region is also
clearly identifiable. The contours of yeast cells were smooth
and their cytoplasm was non-segregated. Surrounding the
outer surface of the cell wall, fimbriae could be clearly seen.
Immediately under the cell membrane, there are numerous
finger-like invaginations. Inside yeast cells, the nucleus and
vacuoles were round or oval with smooth contours. Numerous
mitochondria were often observed inside the cytoplasm.
Occasionally some yeast and bacterial cells appeared dented
and displayed condensed cytoplasm. Yeast cells frozen in
phosphate buffered saline or 0.3 M sucrose showed similar
preservation to cells frozen in pure water.
With C. elegans, well-preserved adults, larvae, and
embryos were observed next to damaged individuals.
Fig. 1. Clamp-sealed capillary tubes. 1(A) Tubes sealed with pliers. Scale bar: 1 div = 1 mm. 1(B) Higher magnification showing the sealed end is
between 1 and 1.5 mm long. 1(C) Cryo-SEM image of the clamp-sealed end of a copper tube filled with water and frozen in liquid nitrogen observed at a
temperature < −100◦ C. There is no detectable leakage. The slit (white arrows) is less than1 μm wide. Scale bar = 20 μm.
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J.L.M. LEUNISSEN AND H. YI
Fig. 2. Examples of cryosubstituted bacteria and yeast cells. The specimens were frozen in a pure water environment and no cryoprotectants were added.
(A), (B) Thermophilic bacteria Anoxybacillus flavithermus, the capsule surrounding the bacteria is well preserved. Scale bars: A = 2.5 μm; B = 0.5 μm.
(C), (D) Yeast cells showing cytoplasm with vacuole, nucleus, mitochondria and vesicles below the plasma membrane with invaginations. D shows an
unidentified vesicle cluster. Scale bars: C = 1 μm; D = 0.8 μm.
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Journal compilation SELF-PRESSURIZED RAPID FREEZING (SPRF)
29
Fig. 3. Examples of cryosubstituted C. elegans frozen by plunging tubes into liquid propane at −180◦ C. The specimens were frozen in a pure water
environment and no cryoprotectants were added. C is a higher magnification of the boxed-in area in A. Scale bars: A = 2 μm, B = 1 μm,
C = 0.5 μm.
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J.L.M. LEUNISSEN AND H. YI
Fig. 4. Examples of cryosubstituted C. elegans frozen by plunging tubes into liquid propane at −120◦ C. The specimens were frozen in a pure water
environment and no cryoprotectants were added. C is a higher magnification of the boxed-in area in A. Scale bars: A = 2 μm, B = 2 μm, C = 0.5 μm.
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Journal compilation SELF-PRESSURIZED RAPID FREEZING (SPRF)
31
Fig. 5. High magnification details of cryosubstituted Anoxybacillus flavithermus (5A), Saccharomyces cerevisiae (5B) and Caenorhabditis elegans (5C)
illustrating the absence of segregation patterns. Scale bars 0.1 μm.
Occasionally, especially in adult worms, heterogeneous
preservation quality was evident within the same individuals.
Nevertheless, excellent ultrastructural preservation was often
seen throughout the entire adult worms. Within wellpreserved C. elegans, all cell and tissue types showed normal,
well-defined ultrastructure.
With C. elegans percentages of well-preserved specimens
were on average approximately 50% whereas sections of
yeast cells showed often close to 100% well-preserved cells.
High magnification images in Fig. 5 show the absence
of segregation patterns in the cytoplasm of well-preserved
specimens. Variation in quality of the cryopreservation was
the highest with bacterial cells. There was variation in
preservation quality from tube to tube for all specimens.
Adequate preservation could be achieved with all three
types of capillary sizes but no quantitative evaluation was
done.
We also tested freezing down to a temperature closer to the
point of maximum supercooling, using propane at −120◦ C.
The ultrastructure is comparable to that achieved with liquid
nitrogen or liquid propane at −180◦ C (Fig. 4). Freezing in open
tubes always resulted in severe damage by ice crystals (data
not shown).
The quality of the ultrastructural preservation in SPRF is
the result of freezing in a closed container that limits the
expansion of the specimen’s water content. Numerous studies
have established that with high freezing speeds, obtained by
plunge freezing or metal mirror methods a superficial layer of a
few micrometres can be frozen without detectable ice damage.
However, ultrafast freezing alone cannot account for the
quality of ultrastructural preservation in SPRF, because little
difference was observed between freezing in liquid nitrogen
and liquid propane. High cooling rates per se also would not
explain the preservation of adult nematodes with dimensions
of 50–100 μm in width and several hundreds of micrometres
long. Because freezing in open capillaries results in ice crystal
formation and destruction of ultrastructure in all cells, it is
logical to conclude that the preservation achieved in SPRF
is the result of pressure build-up by cooling a water-based
suspension in a confined space.
Discussion
Possible mechanisms for pressure build-up
The SPRF method was first introduced at the Microscopy
& Microanalysis Meeting 2007 (Leunissen & Yi, 2007).
Pressure build-up as the result of water freezing is a common
phenomenon, for example, water pipes bursting in winter.
Additional experiments have been conducted to explore the
potential of SPRF and to attain a better understanding of
underlying mechanisms.
Pressure and the quality of the ultrastructural preservation
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J.L.M. LEUNISSEN AND H. YI
Upon lowering the temperature at ambient pressure water
will either supercool or freeze into hexagonal ice (Ice Ih). Either
or both of these physical events could be responsible for the
pressure build-up.
Supercooling is the phenomenon in which water or a
solution cools below the melting point without a phase
transition to solid ice. This condition is thermodynamically
metastable and only certain precautions will prevent
supercooled water from solidifying. At ambient pressure the
minimum temperature for supercooling is approximately
−42◦ C, but under 210 MPa pressure supercooling can take
place down to temperatures as low as −90◦ C. Water expands
during supercooling as a function of temperature as an
increasing number of water molecules adopt a more open
intermolecular structure in preparation for solidification.
Densities of 0.9999 g cm−3 and 0.9775 g cm−3 have been
reported for liquid water at 0◦ C and −34◦ C, respectively
(Hare & Sorensen, 1987). In a confined space this tendency
for density change results in an increase in pressure. It is
unknown whether the pressure increase during supercooling
in the temperature range between −22◦ C and −90◦ C would
be adequate to provide for the ultrastructural preservation
illustrated in this study. It is however intriguing to note that
with increasing pressure the melting point as well as the
supercooling temperature drop, which would favor structural
cryopreservation.
In water solutions containing particulate impurities, or in
water enclosed in rough-surfaced containers, heterogeneous
ice nucleation prevails and ice crystallization eventuates soon
after the melting point is reached during cooling. The lower the
heat extraction rate, the more likely is this scenario in solutions
containing living biological specimens. In a confined space at
temperatures between 0◦ C and −22◦ C, the compressibility
of liquid water allows for a defined amount of water to
expand into low-density Ice Ih. Whereas the amount of Ice Ih
formed progresses with cooling, the remaining liquid fraction
of water becomes more compressed and its density increases.
Hayakawa et al., (1998) measured the relationship between
lowering temperature and pressure build-up. They measured
pressures of 60 MPa at −5◦ C, 103 MPa at −10◦ C and 140 MPa
at −15◦ C. These values are in agreement with the melting
point/pressure diagram for water. The formation of Ice Ih is
increasingly counteracted as the pressure mounts, reaching a
maximum of approximately 210 MPa at −22◦ C (Hayashi &
Nishimura, 2002; Hayashi et al., 2002).
The approximate fraction (F) of the original water mass
that needs to be converted to Ice Ih to generate 210 MPa
pressure at −22◦ C in a confined space can be calculated from
the density of water before cooling (d w0 ), the density of the
pressurized water (d wp ) and the density of hexagonal ice (d i ) as
follows:
F=
d i (d wp − d w0 )
d w0 (d wp − d i )
The density of the compressed water can be calculated from
d wp =
d w0
1 − P · β
where P represents the pressure increase and β the
compressibility of water. Both water compressibility and Ice Ih
density (d i ) are temperature and pressure dependent. Values
for β vary between approximately 5.1 × 10−10 Pa−1 at
0◦ C and atmospheric pressure and 3.3 × 10−10 Pa−1 at
−20◦ C and 200 MPa (Kanno & Angell, 1979; DeBenedetti,
2003). Ice Ih density (d i ) values vary between 0.9167 at
0◦ C and at atmospheric pressure and 0.928 at −22◦ C and
210 MPa (Eisenberg & Kauzmann, 1969; Marion and
Jakubowski, 2004). When the pressure reaches 210 MPa, the
density of compressed water would increase to approximately
1.08 g cm−3 , therefore F would equal about 0.5, which means
50% of the original water mass would have been converted
to Ice Ih. Thermodynamic calculations for a theoretical model
system led Rubinsky et al. (2005) to similar values.
Pressure may be influenced by shrinkage of the capillary
during cooling. The temperature dependent change in cavity
volume of the specimen container (V) is determined as
V = 3 · α · T · V
where α is the thermal linear expansion coefficient (for copper
α = 16.5 × 10−6 K−1 ), T the drop in temperature (K),
and V the initial volume of the cavity. With a temperature
drop of approximately 45◦ (from room temperature to −22◦ C
when the pressure has reached a maximum) this would imply
V = 0.2%. This volume change is small compared to the
volume increase contributed by the formation of Ice Ih.
Retaining the pressure
For the pressure to be retained it is a prerequisite to use
containers that will not significantly deform. The tubes we
used to explore the principle of SPRF are identical to those used
as specimen carriers in the Leica EmPact HPF instruments.
They have proven adequate to resist bursting while being
pressurized to 200 MPa at room temperature immediately
before freezing. Plastic as well as elastic deformation can be
tolerated in this case as it does not affect the externally applied
and buffered pressure. The pressure tolerance requirements
for capillaries used in SPRF are more strict: both elastic
and plastic deformation would result in an increase in the
volume occupied by Ice Ih and a decrease in the volume
that could be adequately pressurized. A favorable factor in
the SPRF technique is that the tube walls cool before and
while the pressure builds up. This further increases the
capillary’s resistance to deformation. If all the enclosed water
were to freeze into Ice Ih, and shrinkage of the capillary
as a result of the cooling would be taken into account, the
volume increase would result in an outer diameter increase
of about 13 μm assuming there is no increase in tube length.
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No such an increase in the tube diameter was observed in
cryo-SEM.
The clamped tube ends are flattened and are therefore weak
spots. To prevent the clamped tube ends from leaking or
bursting the tubes were submerged parallel to the cryogen
surface. This ensures that the two clamped ends of the tubes
cool simultaneously. Presumably the micrometer-thin layer
of liquid between the flattened tube walls will freeze first
and therefore seal the containers preventing leakage from
the clamped ends. This was demonstrated using capillaries
filled with an aqueous Toluidine blue solution that facilitates
detection of leaks. When such specimens were plunge cooled
horizontally into liquid nitrogen or liquid propane, sealed
specimen holders showed no visible leaks through the clamped
ends of the capillary tubes.
The physical state of water in the capillary tubes after cooling
With regard to the physical state of water and ice in
SPRF, parallels to HPF seem likely. However, there are also
differences. In the HPF technique the pressure is applied
prior to cooling. As a result, the specimen temperature is
likely to increase by 2–10 degrees (Dumay et al. 2006)
immediately before applying the cryogen. In SPRF the pressure
is built up gradually during cooling and at some point
pressures are expected to be similar to the ones applied
in HPF. A further and remarkable difference with HPF
is the absence of cryoprotectants in the SPRF samples.
Lepault et al. (1997) reported that the presence and
concentration of cryoprotectant while cooling at similar
cooling speeds influences whether amorphous or hexagonal
or cubic crystalline ice is formed illustrating the influence of
cryoprotectants on ice formation.
We have not investigated the physical state of water within
the capillary tubes in the SPRF approach, but we anticipate
that the average density of the enclosed water must still be
close to 1 g cm−3 , because there is no significant change in the
capillary tube dimensions. Assuming that Ice Ih is responsible
for the pressure build-up, and also assuming that about 50% of
the water mass needs to be converted to Ice Ih for that purpose,
then the remaining 50% of the water mass must have an
average density of approximately 1.08. Likewise, the density of
water would also increase to about 1.08 in HPF when pressure
of 200–210 MPa is applied at room temperature. This implies
that the water volume would be reduced by approximately
9%.
Which polymorphs and/or polyamorphs of ice are formed
in the pressurized capillary tubes will depend on cooling speed,
specimen composition, cryogen temperature and pressure.
From the phase/pressure diagram for water it appears that
at the indicated pressure of 210 MPa in addition to Ice Ih
and Ic Ice II, III, IX are possible polymorphs as well as LDA
and possibly HDA (low- and high-density amorphous ice,
respectively). Their densities are summarized in Table 1.
33
Table 1. Typical densities of crystalline and acrystalline ice forms.
Ice poly(a)morph
Density (g cm−3 )
Ice Ih
Ice Ic
Ice II
Ice III
Ice IX
LDA
HDA
0.92
0.92
1.17
1.14
1.16
0.94
1.17–1.19
Using diffraction methods polymorphs Ih, Ic (cubic ice) and
III as well as vitreous ice have been illustrated to exist in HPF
specimens (Erk et al., 1998; Richter, 1994; Dubochet, 1995;
Lepault et al., 1997). Based on density and the absence of
a detectable crystalline structure, vitreous ice most closely
resembles the liquid state of water. For that reason it is the
preferred form of ‘ice’ and is thought to be the least damaging
to structures. Vitrification results following a special form
of supercooling in which heat extraction is faster than the
latent heat release. As a result crystallization does not occur.
This has been referred to as hyperquenching or hypercooling
(Akyurt et al., 2002). As probabilistic rather than kinetic
events (Debenedetti, 2003), the chance for suspensions of
biological organisms to become supercooled, hypercooled
and vitrified is higher as freezing speeds increase (Franks,
2003).
In addition to vitreous ice, other forms of amorphous ice
have been described which are differentiated by their density
and sometimes by the way they are formed. Of those, only
LDA and HDA can exist under the temperature and pressure
conditions described. It should be pointed out that the names
LDA and vitreous ice are often used interchangeably and
despite observed differences they have been proposed to be
considered identical (Debenedetti, 2003). For the purpose of
ultrastructural preservation their characteristics are at least
very similar. Both have a density of 0.94 g cm−3 , which is
close to the density of Ice Ih and Ic. HDA ice is an amorphous
ice with a density of 1.17. The densities change slightly with
temperature and pressure.
The densities of the different forms of ice are relevant for their
potential occurrence in SPRF specimens. First, at a pressure
of approximately 200 MPa the density of the liquid water is
likely to be 1.08 as calculated earlier. Consequentially, the
average density of the resulting solid water must also be 1.08,
provided the volume of the pressurized water does not change.
But so far no crystalline or a-crystalline form of water has been
described with such a density. Hence it follows there must be
a mixture of densities and thus a mixture of different ices. One
might speculate that when ice with a density higher than the
compressed water is formed, this immediately results in a slight
drop of pressure compensated for by a further formation of lowdensity ice, whether crystalline or amorphous. With the state
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of the art we suggest that ice polymorphs and polyamorphs
similar to those found in HPF specimens constitute this blend.
However, although in-depth research is conducted into water
poly(a)morphism new and often controversial views regarding
supercooling and vitrification are presented on a regular basis
(Bower et al., 2002; Angell, 2004; Kohl et al., 2005; Szobota
& Rubinsky, 2006).
Second, in both SPRF and HPF a phase shift from water
to amorphous or crystalline ice is accompanied by a density
shift. When water with a density of 1.08 vitrifies to LDA with
a density of 0.94 an initial density increase is followed by
a density decrease in a short time span, resulting in a net
density shift of close to 15%. It can not be excluded that
these density shifts cause changes in biological specimens,
calling for caution when attempting to draw conclusions from
observations on natively frozen specimens.
Evaluation of ultrastructural preservation
The structural preservation achieved by SPRF of the yeast and
C. elegans specimens we used as test samples is in line with
what has been described using, e.g. HPF followed by freeze
substitution (Müller-Reichert et al., 2003, 2008; Murray,
2008) to establish the potential for ultrastructural research as
well as immuno-detection studies. The structural preservation
in SPRF, however, was achieved after freezing specimens in a
pure water environment. This condition was chosen to exclude
any possibility that compounds present in buffers or growth
media may act as a cryoprotectant, illustrating the essential
potential of SPRF. Yeast cells frozen in buffer or 0.3 M sucrose
showed a similar quality of preservation.
Specimens that were cryofixed using the SPRF method
and cryosubstituted using established protocols show many
well-preserved cells and organisms without visible signs of
ice crystal damage as revealed by the absence of segregation
patterns. This observation is encouraging because in most
cases the specimens were frozen from distilled or tap water
or a simple physiological buffer. Beside well-preserved areas,
there are areas showing more or less crystal damage caused by
the formation of low-density ice inside the capillary, leading to
pressure build up. As long as biological specimens are evenly
distributed throughout the capillary tube it is plausible that
on average 50% of the structures will be preserved in a way
similar to HPF specimens. This percentage was observed for
C. elegans, but for yeast cells the percentage was higher.
When ice is formed the ionic strength of the remaining
solution increases by exclusion of dissolved substances from
the solidifying water. Assuming 50–55% of the water changed
into hexagonal ice there is on average a doubling in ionic
strength in the remaining liquid. Considering the very brief
period of time specimens may be exposed to this increased
ionic strength before becoming cryofixed the effect may not
be significant, although this remains to be established. Ion
distributions will change with any phase change of the water
and will thus be best preserved in vitreous ice or when ice
crystals are small.
The role of the cryogen/cooling speed
In HPF, minimum required cooling speeds are expected to be
in the order of 1000 K s−1 . With this approach specimens as
thick as 200–600 μm may be adequately cryofixed (Dahl &
Staehelin, 1989; Studer et al, 1989; Vanhecke et al, 2008). It
is not very likely that these cooling speeds are realized inside
the specimen with liquid nitrogen as the cryogen in SPRF.
Studer et al. (1995) and Shimoni & Müller (1998) describe
how the cooling speed towards the centre of the specimen is
limited by conductive heat transfer and that this is the reason
for the limitations of adequate freezing in any cryo technique.
In these publications it was argued that using more efficient
cooling agents will not significantly increase the depth of
proper freezing in HPF, as the heat conductivity of water is
the rate-limiting factor.
The degree of preservation of the test specimens in SPRF
can not be easily explained by the theories and observations
illustrated in the publications mentioned above. The cooling
speed achieved with liquid nitrogen in SPRF is certainly lower
than that achieved in HPF instruments because plunging does
not avoid the Leidenfrost phenomenon the way it is avoided
in spraying liquid nitrogen under elevated pressure onto a
specimen. Heat extraction will be higher with propane, but the
quality of the ultrastructural preservation is not significantly
different between the two cryogens. SPRF specimens frozen in
liquid propane at −120◦ C showed preservation similar to that
of specimens frozen at −180◦ C in propane or in liquid nitrogen.
This may open possibilities for the application of hitherto less
elaborately explored cryogens with higher melting points,
such as ethanol, methanol and 1-propanol.
Prospects for further development of SPRF
The data presented demonstrate the usability of SPRF to obtain
ultrastructural preservation in biological specimens, even
when frozen in a pure water environment. Experiments are
underway to investigate how the principles behind SPRF can
be further established and how the efficiency of preservation in
SPRF can be improved. Some of these experiments are aimed
at exploring the suitability of cryoprotectant to improve the
percentage of well-preserved specimens as well as the potential
of larger tubes for the cryofixation of tissue by SPRF.
Acknowledgements
The authors are greatly indebted to Reinhard Lihl, Paul
Wurzinger and Ian Lamswood for stimulating discussions and
continuous support, to Dr. Howard Rees for critically screening
the text and to Prof. Peter Elbers for continuous support and
positive criticism.
C 2009 The Authors
C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35
Journal compilation SELF-PRESSURIZED RAPID FREEZING (SPRF)
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C 2009 The Authors
C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35
Journal compilation