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 freezing in the presence of a cryoprotectant, with high heat extraction rates, and under hyperbaric pressure. A method is described for the cryofixation of For ultrastructural and immunocytochemical biological specimens for ultrastructural analysis and investigations, by far the largest share of all cryo-based immunocytochemical detection studies. The method employs specimen preparations, adequate cell and tissue cryofixation plunge freezing of specimens in a sealed capillary tube may be achieved by applying cryoprotectants (e.g. Tokuyasu, into a cryogen such as liquid propane or liquid nitrogen. 1973; Franks, 1977; Gilkey & Staehelin, 1986). High cooling Using this method a number of single-cell test specimens rates (>105◦Cs−1) allow vitrification of thin layers only were well preserved. Also multicellular organisms, such as (Dubochet & McDowall, 1981), which is not always sufficient Caenorhabditis elegans, could be frozen adequately in low ionic for general ultrastructural and immunocytochemical strength media or even in . The preservation of these detection studies. High-pressure freezing (HPF) as presented unprotected specimens is comparable to that achieved with by Moor & Riehle (1968) is a technique that allows for high-pressure freezing in the presence of cryoprotectant. The a depth of 100–300 μm from the specimen surface to be results are explained by the fact that cooling of water in a frozen without detectable crystallization damage (Dahl & confined space below the melting point gives rise to pressure Staehelin, 1989; Studer et al., 1989; Vanhecke et al., 2008). build-up, which may originate from the conversion of a At 200–210 MPa pressure high cooling rates are no longer a fraction of the water content into low-density hexagonal ice prime requirement. All currently available HPF instruments and/or expansion of water during supercooling. Calculations use liquid nitrogen as the cryogen. indicate the pressure may be similar in magnitude to that HPF has become an accepted technique for the structural applied in high-pressure freezing. Because the specimens are preservation of pro- and eukaryotic cells, multicellular plunge cooled, suitable cryogens are not limited to liquid organisms and tissues, allowing further processing with nitrogen. It is shown that a range of cryogens and cryogen cryosubstitution (Monaghan et al., 1998; Hawes et al., temperatures can be used successfully. Because the pressure 2007; Buser & Walther, 2008; Triffo et al., 2008), freeze is generated inside the specimen holders as a result of the fracturing (Dahl & Staehelin, 1989; Walther, 2003) and cooling rather than applied from an external source as in cryoultramicrotomy (van Donselaar et al., 2007). Despite high-pressure freezing, the technique has been referred to as its suitability to freeze unprotected specimens, in many self-pressurized rapid freezing. published applications HPF is combined with the use of a non- penetrating cryoprotectant such as dextran or serum albumin to further support cryopreservation (Dubochet, 1995). In Introduction the absence of cryopreservatives crystalline ice is formed in Physical fixation of biological specimens is the basic principle the extracellular space, which affects preservation (Erk et al., underlying a range of specimen preparation methods in 1998; Dubochet, 1995). electron microscopy aimed at preserving ultrastructure, HPF is based on the principle of Le Chatelier and molecular functionality and recognition, as well as electrolyte Braun which postulates that if a system at thermodynamic distribution. Several methods have been developed involving 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 Correspondence to: Jan L.M. Leunissen. E-mail: [email protected] an externally applied pressure of between 200 and 210 MPa

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society 26 J.L.M. LEUNISSEN AND H. YI prevents water from expanding into low-density ice upon ensuring a tight fit. The micropipette volume was set to a cooling. few microlitres in excess of the tube capacity (1.54 μL) to The present manuscript introduces the self-pressurized allow for slight overfilling of the capillary (4–5 μL). The open rapid freezing (SPRF) method based on the same principle end of the capillary was inserted into the specimen suspension of Le Chatelier and Braun. It uses the tendency for water and the suspension was drawn into the tube slowly using inside the specimen container to expand upon cooling, thereby the micropipette action, preventing air bubbles from forming generating pressure intrinsically instead of using an external inside the capillary. If necessary the process was repeated. hydraulic system. This pressure is likely to be the result of two processes: ice crystallization and/or supercooling which Capillary tube sealing is the phenomenon that water remains liquid at temperatures well below the melting point of ice. The ambient pressure The filled capillary tube, while mounted in the micropipette tip, polymorphs of ice as well as supercooled water have a lower was withdrawn from the suspension. The open end was sealed density than water at 0◦C. Therefore, upon cooling a specimen by clamping a length of about 1–1.5 mm shut using needle- that is enclosed in a confined space below 0◦C pressure will be nose pliers with flat jaws. Next, while pressing the pipette generated. In practice, pressures comparable to those applied piston down, the capillary was removed from the micropipette in HPF can be generated in this way (Hayashi et al., 2002). tip and the open capillary end sealed by tightly clamping a In this manuscript, we report how this phenomenon can be length of about 1–1.5 mm shut using the pliers. For controls, exploited for ultrastructural preservation studies. tubes were filled as described but not sealed.

Cryoprocessing Materials and methods Sealed capillaries containing specimens were either dipped Specimens manually or plunged into liquid nitrogen or liquid propane (−180◦Cor−120◦C) or melting isopentane using either an Caenorhabditis elegans (C. elegans) nematodes were a kind gift of EMS Plunge 002 (EMS, Hatfield PA, USA), a Leica/Reichert Dr. Guy Benian, Dept. of Pathology and Laboratory Medicine, KF-80 or a Leica CPC (both Leica, Austria) as follows. Emory University, Atlanta. Bacterial cultures (Anoxybacillus Capillaries, held with fine forceps, were inserted into the flavithermus and Geobacillus stearothermophilus)werekindly cryogen horizontally to assure that the clamped ends were provided by Hannah Heinrich, MSc (Dept. of Biochemistry, inserted simultaneously. Specimens were left in cryogen for a University of Otago). Yeast cells (Saccharomyces cerevisiae) minimum of 15 s after which they were stored under liquid were kindly provided by Dr. Paul Doetsch and Dr. Natalya nitrogen awaiting further processing. Degtyareva, Dept. of Biochemistry, Emory University, Atlanta. Bacteria, yeast cells and nematodes were collected and frozen Further processing and freeze substitution in distilled water. Alternatively yeast cells were collected and frozen in 10 mM phosphate buffer, 145 mM NaCl, pH 7.4 or in Capillaries were cut under liquid nitrogen into approximately 0.3 M sucrose solution in water. 1–2-mm-longsegmentswithapre-cooledcapillarytubecutter (DT-001, Wujiang Dunnex Tools Co. Ltd, Suzhou, China). Specimen holders Using a capillary tube cutter prevents the capillary from collapsing during cutting. Alternatively they were cut into Capillary copper tubes 16 mm in length and with an outer 6-mm-long segments and sliced open under liquid nitrogen diameter (o.d.) of 0.65 mm and an inner diameter (i.d.) of to expose the contents using the Leica HPF cryotools (Leica 0.35 mm were obtained from Leica Austria (Leica part number product package 16706855). Capillary segments containing 16706871) (Leica, Vienna, Austria). These capillary tubes specimen were then transferred under liquid nitrogen into a contain 1.54 μL when they are fully filled with specimen. cryo vial that contained frozen substitution medium. Samples In addition copper capillaries with nominal dimensions of were freeze-substituted (van Harreveld & Crowell, 1964) in a 0.8 mm o.d., 0.3 mm i.d. and 1.3 mm o.d., 0.4 mm i.d. Leica AFS automatic freeze substitution apparatus. Segments (Albion Alloys Ltd, Bournemouth, UK) were used. Capillary cut from two capillary tubes were sometimes pooled to ensure tubes were pre-cleaned by sonication in 0.1% Triton-X-100 to sufficient specimen volume for further processing. hydrophilize the interior surface to prevent air bubbles from Substitution took place in acetone, containing 2% OsO and sticking and rinsed in distilled water before use. 4 0.1% uranyl acetate. Typically, specimens were kept at −90◦C for 72 h, occasionally turning the substitution vials 360◦ to Capillary tube mounting and specimen loading make sure dissolved ice did not accumulate at the bottom Capillary tubes were inserted into a disposable pipette tip of the vials containing the substitution solution. Hereafter mounted on a 0–20 μL micropipette. Part of the pipette tip the temperature was raised at a rate of 2◦C/hr to −60◦C, was cut off to facilitate mounting of the capillaries while −30◦Cand0◦C and kept at each level for 8 h in the original

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 SELF-PRESSURIZED RAPID FREEZING (SPRF) 27 substitution medium. Vials containing specimen were then from the values at room temperature. Cryo-SEM Scanning removed from the AFS and left at 4◦C for 1 h before being Electron Microscopy examination revealed no leakage from brought to room temperature for subsequent processing. the clamped ends of water-filled frozen capillaries (Fig. 1). At Substitution medium containing specimen material was high magnifications it appears that the slit width is about transferred to an Eppendorf tube and centrifuged. Part of the 1 μm or less. No signs of leakage were apparent as a result of substitutionmediumwasremovedwithcaretoavoidspecimen pressure developed inside the capillaries. By contrast, dipping material at the tip of the tube from being disturbed. Acetone the tubes perpendicular to the cryogen surface often resulted (100%) was added to the capillary segments remaining in the in leaking of the top seal. original cryo vial. Any sample material remaining inside the Open capillaries clearly showed expansion of the liquid from capillary segments was pushed out using an eye lash probe or inside the tube upon cooling such specimens in cryogen. 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 Ultrastructural preservation Eppendorf tube for centrifugation. Freeze-substituted bacteria, yeast cells as well as C. elegans Specimens were rinsed at room temperature three times nematodes were observed after freezing in clamp-sealed with 100% acetone and then embedded in epoxy resin capillary copper tubes using either liquid nitrogen at −196◦C (Quetol or Spurr), ultrathin sections were post-contrasted or liquid propane at −180◦Cor−120◦C (Figs 2–5). using aqueous uranyl acetate for 10–15 min and lead citrate Thermophilic bacteria Anoxybacillus flavithermus and for 8–10 min at room temperature. Geobacillus stearothermophilus showed typical ultrastructure of gram-positivebacteriawithclearlydefinedplasmamembrane, peptidoglycan cell wall and polysaccharide capsule. Within Results these bacteria the cytoplasm appeared with no apparent segregation. The most distinct structure inside the bacteria The effect of freezing on capillary tubes was ribosomes. On occasion the nucleoid region is also The success of SPRF depends on generating pressure inside clearly identifiable. The contours of yeast cells were smooth the clamp-sealed specimen capillary tube and holding that and their cytoplasm was non-segregated. Surrounding the pressure for the time it takes to cryopreserve the specimen. outer surface of the cell wall, fimbriae could be clearly seen. Tests were therefore carried out to verify that the sealed Immediately under the cell membrane, there are numerous ends of the tubes (Fig. 1) would not leak and that finger-like invaginations. Inside yeast cells, the nucleus and there was no deformation of the tubes during the cooling vacuoleswereroundorovalwithsmoothcontours.Numerous process. Cooling by dipping or plunging in liquid nitrogen mitochondria were often observed inside the cytoplasm. or propane, where the clamp-sealed capillary is inserted Occasionally some yeast and bacterial cells appeared dented parallel to the cryogen surface, resulted in no noticeable and displayed condensed cytoplasm. Yeast cells frozen in deformation or bursting of the capillaries. The outer diameter phosphate buffered saline or 0.3 M sucrose showed similar of both empty and distilled water-filled capillaries before preservation to cells frozen in pure water. and after freezing as observed in cryo-SEM at a specimen With C. elegans, well-preserved adults, larvae, and stage temperature of −180◦C is not noticeably different 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.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 28 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.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 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.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 30 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.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 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 Additional experiments have been conducted to explore the preservation quality was evident within the same individuals. potential of SPRF and to attain a better understanding of Nevertheless, excellent ultrastructural preservation was often underlying mechanisms. seen throughout the entire adult worms. Within well- preserved C. elegans, all cell and tissue types showed normal, Pressure and the quality of the ultrastructural preservation well-defined ultrastructure. With C. elegans percentages of well-preserved specimens The quality of the ultrastructural preservation in SPRF is were on average approximately 50% whereas sections of the result of freezing in a closed container that limits the yeast cells showed often close to 100% well-preserved cells. expansion of the specimen’s water content. Numerous studies High magnification images in Fig. 5 show the absence have established that with high freezing speeds, obtained by of segregation patterns in the cytoplasm of well-preserved plunge freezing or metal mirror methods a superficial layer of a specimens. Variation in quality of the cryopreservation was few micrometres can be frozen without detectable ice damage. the highest with bacterial cells. There was variation in However, ultrafast freezing alone cannot account for the preservation quality from tube to tube for all specimens. quality of ultrastructural preservation in SPRF, because little Adequate preservation could be achieved with all three difference was observed between freezing in liquid nitrogen types of capillary sizes but no quantitative evaluation was and liquid propane. High cooling rates per se also would not done. explain the preservation of adult nematodes with dimensions We also tested freezing down to a temperature closer to the of 50–100 μm in width and several hundreds of micrometres point of maximum supercooling, using propane at −120◦C. long. Because freezing in open capillaries results in ice crystal The ultrastructure is comparable to that achieved with liquid formation and destruction of ultrastructure in all cells, it is nitrogen or liquid propane at −180◦C (Fig. 4). Freezing in open logical to conclude that the preservation achieved in SPRF tubes always resulted in severe damage by (data is the result of pressure build-up by cooling a water-based not shown). suspension in a confined space.

Discussion Possible mechanisms for pressure build-up The SPRF method was first introduced at the Microscopy Pressure build-up as the result of water freezing is a common & Microanalysis Meeting 2007 (Leunissen & Yi, 2007). phenomenon, for example, water pipes bursting in winter.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 32 J.L.M. LEUNISSEN AND H. YI

Upon lowering the temperature at ambient pressure water The density of the compressed water can be calculated from will either supercool or freeze into hexagonal ice (). Either dw0 or both of these physical events could be responsible for the dwp = 1 − P · β pressure build-up. Supercooling is the phenomenon in which water or a where P represents the pressure increase and β the solution cools below the melting point without a phase compressibility of water. Both water compressibility and Ice Ih transition to solid ice. This condition is thermodynamically density (di) are temperature and pressure dependent. Values − − metastable and only certain precautions will prevent for β vary between approximately 5.1 × 10 10 Pa 1 at ◦ − − supercooled water from solidifying. At ambient pressure the 0 C and atmospheric pressure and 3.3 × 10 10 Pa 1 at ◦ minimum temperature for supercooling is approximately −20 C and 200 MPa (Kanno & Angell, 1979; DeBenedetti, ◦ −42 C, but under 210 MPa pressure supercooling can take 2003). Ice Ih density (di) values vary between 0.9167 at ◦ ◦ place down to temperatures as low as −90◦C. Water expands 0 C and at atmospheric pressure and 0.928 at −22 Cand during supercooling as a function of temperature as an 210 MPa (Eisenberg & Kauzmann, 1969; Marion and increasing number of water molecules adopt a more open Jakubowski, 2004). When the pressure reaches 210 MPa, the intermolecular structure in preparation for solidification. density of compressed water would increase to approximately − Densities of 0.9999 g cm−3 and 0.9775 g cm−3 have been 1.08 g cm 3, therefore F would equal about 0.5, which means reported for liquid water at 0◦Cand−34◦C, respectively 50% of the original water mass would have been converted (Hare & Sorensen, 1987). In a confined space this tendency to Ice Ih. Thermodynamic calculations for a theoretical model for density change results in an increase in pressure. It is system led Rubinsky et al. (2005) to similar values. unknown whether the pressure increase during supercooling Pressure may be influenced by shrinkage of the capillary in the temperature range between −22◦Cand−90◦C would during cooling. The temperature dependent change in cavity be adequate to provide for the ultrastructural preservation volume of the specimen container (V) is determined as illustrated in this study. It is however intriguing to note that V = 3 · α · T · V with increasing pressure the melting point as well as the supercooling temperature drop, which would favor structural where α is the thermal linear expansion coefficient (for copper cryopreservation. α = 16.5 × 10−6 K−1), T the drop in temperature (K), In water solutions containing particulate impurities, or in and V the initial volume of the cavity. With a temperature water enclosed in rough-surfaced containers, heterogeneous drop of approximately 45◦ (from room temperature to −22◦C ice nucleation prevails and ice crystallization eventuates soon when the pressure has reached a maximum) this would imply after the melting point is reached during cooling. The lower the V = 0.2%. This volume change is small compared to the heatextractionrate,themorelikelyisthisscenarioinsolutions volume increase contributed by the formation of Ice Ih. containing living biological specimens. In a confined space at temperatures between 0◦Cand−22◦C, the compressibility Retaining the pressure of liquid water allows for a defined amount of water to expand into low-density Ice Ih. Whereas the amount of Ice Ih For the pressure to be retained it is a prerequisite to use formed progresses with cooling, the remaining liquid fraction containers that will not significantly deform. The tubes we of water becomes more compressed and its density increases. used to explore the principle of SPRF are identical to those used Hayakawa et al., (1998) measured the relationship between as specimen carriers in the Leica EmPact HPF instruments. lowering temperature and pressure build-up. They measured They have proven adequate to resist bursting while being pressuresof60MPaat−5◦C,103MPaat−10◦Cand140MPa pressurized to 200 MPa at room temperature immediately at −15◦C. These values are in agreement with the melting before freezing. Plastic as well as elastic deformation can be point/pressure diagram for water. The formation of Ice Ih is tolerated in this case as it does not affect the externally applied increasingly counteracted as the pressure mounts, reaching a and buffered pressure. The pressure tolerance requirements maximum of approximately 210 MPa at −22◦C (Hayashi & for capillaries used in SPRF are more strict: both elastic Nishimura, 2002; Hayashi et al., 2002). and plastic deformation would result in an increase in the The approximate fraction (F) of the original water mass volume occupied by Ice Ih and a decrease in the volume that needs to be converted to Ice Ih to generate 210 MPa that could be adequately pressurized. A favorable factor in pressure at −22◦C in a confined space can be calculated from the SPRF technique is that the tube walls cool before and the density of water before cooling (dw0), the density of the while the pressure builds up. This further increases the pressurized water (dwp) and the density of hexagonal ice (di)as capillary’s resistance to deformation. If all the enclosed water follows: were to freeze into Ice Ih, and shrinkage of the capillary as a result of the cooling would be taken into account, the di(dwp − dw0) volume increase would result in an outer diameter increase F = dw0(dwp − di) of about 13 μm assuming there is no increase in tube length.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 SELF-PRESSURIZED RAPID FREEZING (SPRF) 33

No such an increase in the tube diameter was observed in Table 1. Typical densities of crystalline and acrystalline ice forms. cryo-SEM. − The clamped tube ends are flattened and are therefore weak Ice poly(a)morph Density (g cm 3) spots. To prevent the clamped tube ends from leaking or Ice Ih 0.92 bursting the tubes were submerged parallel to the cryogen 0.92 surface. This ensures that the two clamped ends of the tubes Ice II 1.17 cool simultaneously. Presumably the micrometer-thin layer Ice III 1.14 of liquid between the flattened tube walls will freeze first Ice IX 1.16 and therefore seal the containers preventing leakage from LDA 0.94 the clamped ends. This was demonstrated using capillaries HDA 1.17–1.19 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 Using diffraction methods polymorphs Ih, Ic (cubic ice) and specimen holders showed no visible leaks through the clamped III as well as vitreous ice have been illustrated to exist in HPF ends of the capillary tubes. 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 The physical state of water in the capillary tubes after cooling resembles the liquid state of water. For that reason it is the With regard to the physical state of water and ice in preferred form of ‘ice’ and is thought to be the least damaging SPRF, parallels to HPF seem likely. However, there are also to structures. Vitrification results following a special form differences. In the HPF technique the pressure is applied of supercooling in which heat extraction is faster than the prior to cooling. As a result, the specimen temperature is latent heat release. As a result crystallization does not occur. likely to increase by 2–10 degrees (Dumay et al. 2006) This has been referred to as hyperquenching or hypercooling immediatelybeforeapplyingthecryogen.InSPRFthepressure (Akyurt et al., 2002). As probabilistic rather than kinetic is built up gradually during cooling and at some point events (Debenedetti, 2003), the chance for suspensions of pressures are expected to be similar to the ones applied biological organisms to become supercooled, hypercooled in HPF. A further and remarkable difference with HPF and vitrified is higher as freezing speeds increase (Franks, is the absence of cryoprotectants in the SPRF samples. 2003). Lepault et al. (1997) reported that the presence and In addition to vitreous ice, other forms of concentration of cryoprotectant while cooling at similar have been described which are differentiated by their density cooling speeds influences whether amorphous or hexagonal and sometimes by the way they are formed. Of those, only or cubic crystalline ice is formed illustrating the influence of LDA and HDA can exist under the temperature and pressure cryoprotectants on ice formation. conditions described. It should be pointed out that the names We have not investigated the physical state of water within LDA and vitreous ice are often used interchangeably and the capillary tubes in the SPRF approach, but we anticipate despite observed differences they have been proposed to be that the average density of the enclosed water must still be considered identical (Debenedetti, 2003). For the purpose of close to 1 g cm−3, because there is no significant change in the ultrastructural preservation their characteristics are at least capillary tube dimensions. Assuming that Ice Ih is responsible very similar. Both have a density of 0.94 g cm−3, which is for the pressure build-up, and also assuming that about 50% of close to the density of Ice Ih and Ic. HDA ice is an amorphous the water mass needs to be converted to Ice Ih for that purpose, ice with a density of 1.17. The densities change slightly with then the remaining 50% of the water mass must have an temperature and pressure. averagedensityofapproximately1.08.Likewise,thedensityof Thedensitiesofthedifferentformsoficearerelevantfortheir water would also increase to about 1.08 in HPF when pressure potential occurrence in SPRF specimens. First, at a pressure of 200–210 MPa is applied at room temperature. This implies of approximately 200 MPa the density of the liquid water is that the water volume would be reduced by approximately likely to be 1.08 as calculated earlier. Consequentially, the 9%. average density of the resulting solid water must also be 1.08, Which polymorphs and/or polyamorphs of ice are formed provided the volume of the pressurized water does not change. in the pressurized capillary tubes will depend on cooling speed, But so far no crystalline or a-crystalline form of water has been specimen composition, cryogen temperature and pressure. described with such a density. Hence it follows there must be From the phase/pressure diagram for water it appears that a mixture of densities and thus a mixture of different . One at the indicated pressure of 210 MPa in addition to Ice Ih might speculate that when ice with a density higher than the and Ic Ice II, III, IX are possible polymorphs as well as LDA compressedwaterisformed,thisimmediatelyresultsinaslight and possibly HDA (low- and high-density amorphous ice, drop of pressure compensated for by a further formation of low- respectively). Their densities are summarized in Table 1. density ice, whether crystalline or amorphous. With the state

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 34 J.L.M. LEUNISSEN AND H. YI of the art we suggest that ice polymorphs and polyamorphs and will thus be best preserved in vitreous ice or when ice similar to those found in HPF specimens constitute this blend. crystals are small. However, although in-depth research is conducted into water poly(a)morphismnewandoftencontroversialviewsregarding supercooling and vitrification are presented on a regular basis The role of the cryogen/cooling speed (Bower et al., 2002; Angell, 2004; Kohl et al., 2005; Szobota In HPF, minimum required cooling speeds are expected to be & Rubinsky, 2006). in the order of 1000 K s−1. With this approach specimens as Second, in both SPRF and HPF a phase shift from water thick as 200–600 μm may be adequately cryofixed (Dahl & to amorphous or crystalline ice is accompanied by a density Staehelin, 1989; Studer et al, 1989; Vanhecke et al, 2008). It shift. When water with a density of 1.08 vitrifies to LDA with is not very likely that these cooling speeds are realized inside a density of 0.94 an initial density increase is followed by the specimen with liquid nitrogen as the cryogen in SPRF. a density decrease in a short time span, resulting in a net Studer et al. (1995) and Shimoni & Muller¨ (1998) describe density shift of close to 15%. It can not be excluded that how the cooling speed towards the centre of the specimen is these density shifts cause changes in biological specimens, limited by conductive heat transfer and that this is the reason calling for caution when attempting to draw conclusions from for the limitations of adequate freezing in any cryo technique. observations on natively frozen specimens. In these publications it was argued that using more efficient cooling agents will not significantly increase the depth of Evaluation of ultrastructural preservation proper freezing in HPF, as the heat conductivity of water is the rate-limiting factor. The structural preservation achieved by SPRF of the yeast and The degree of preservation of the test specimens in SPRF C. elegans specimens we used as test samples is in line with can not be easily explained by the theories and observations what has been described using, e.g. HPF followed by freeze illustrated in the publications mentioned above. The cooling substitution (Muller-Reichert¨ et al., 2003, 2008; Murray, speed achieved with liquid nitrogen in SPRF is certainly lower 2008) to establish the potential for ultrastructural research as than that achieved in HPF instruments because plunging does well as immuno-detection studies. The structural preservation not avoid the Leidenfrost phenomenon the way it is avoided in SPRF, however, was achieved after freezing specimens in a in spraying liquid nitrogen under elevated pressure onto a purewaterenvironment.Thisconditionwaschosentoexclude specimen. Heat extraction will be higher with propane, but the any possibility that compounds present in buffers or growth quality of the ultrastructural preservation is not significantly media may act as a cryoprotectant, illustrating the essential different between the two cryogens. SPRF specimens frozen in potential of SPRF. Yeast cells frozen in buffer or 0.3 M sucrose liquid propane at −120◦C showed preservation similar to that showed a similar quality of preservation. ofspecimensfrozenat−180◦Cinpropaneorinliquidnitrogen. Specimens that were cryofixed using the SPRF method This may open possibilities for the application of hitherto less and cryosubstituted using established protocols show many elaborately explored cryogens with higher melting points, well-preserved cells and organisms without visible signs of such as ethanol, methanol and 1-propanol. 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 Prospects for further development of SPRF or a simple physiological buffer. Beside well-preserved areas, The data presented demonstrate the usability of SPRF to obtain there are areas showing more or less crystal damage caused by ultrastructural preservation in biological specimens, even the formation of low-density ice inside the capillary, leading to when frozen in a pure water environment. Experiments are pressure build up. As long as biological specimens are evenly underway to investigate how the principles behind SPRF can distributed throughout the capillary tube it is plausible that be further established and how the efficiency of preservation in on average 50% of the structures will be preserved in a way SPRF can be improved. Some of these experiments are aimed similar to HPF specimens. This percentage was observed for at exploring the suitability of cryoprotectant to improve the C. elegans, but for yeast cells the percentage was higher. percentage of well-preserved specimens as well as the potential When ice is formed the ionic strength of the remaining of larger tubes for the cryofixation of tissue by SPRF. solution increases by exclusion of dissolved substances from the solidifying water. Assuming 50–55% of the water changed Acknowledgements into hexagonal ice there is on average a doubling in ionic strength in the remaining liquid. Considering the very brief The authors are greatly indebted to Reinhard Lihl, Paul period of time specimens may be exposed to this increased Wurzinger and Ian Lamswood for stimulating discussions and ionic strength before becoming cryofixed the effect may not continuoussupport,toDr.HowardReesforcriticallyscreening be significant, although this remains to be established. Ion the text and to Prof. Peter Elbers for continuous support and distributions will change with any phase change of the water positive criticism.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35 SELF-PRESSURIZED RAPID FREEZING (SPRF) 35

Kohl, I., Bachmann, L., Hallbrucker, A., Mayer, E. & Loerting, T. (2005) References Liquid-like relaxation in hyperquenched water at ≤ 140 K. Phys. Chem. Akyurt, M., Zaki, G. & Habeebullah, B. (2002) Freezing phenomena in ice- Chem. Phys. 7, 3210–3220. water systems. Energy and Conversion Management. 43, 1773–1789. Lepault, J., Bigot, D., Studer, D. & Erk, I. (1997) Freezing of aqueous Angell, C.A. (2004) Amorphous water. Annu. Rev. Phys. Chem. 55, 559– specimens: an X-ray diffraction study. J Microsc. 187(3), 158–166. 583. Leunissen, J.L.M. & Yi, H. (2007) A novel sample freezing method Bower, W.E., Schedgick, D.J. & Kimball Bigelow, L. (2002) Thick glassy (tutorial). Microsc Microanal 13(Suppl 2), 444–445. water by liquid quenching on a diamond wafer. J. Phys. Chem. B, 106, Marion, G.M. & Jakubowski, S.D. (2004) The compressibility of ice to 2.0 4565–4568. kbar. Cold Regions Sci. Tech. 38, 211–218. Buser, C. & Walther, P. (2008) Freeze-substitution: the addition of water Monaghan, P., Perusinghe, N. & Muller,¨ M. (1998) High-pressure freezing to polar solvents enhances the retention of structure and acts at for immunocytochemistry. J. Microsc. 192, 248–258. temperatures around −60 ◦C. J Microsc. 230(Pt 2), 268–277. Moor, H. & Riehle, U. (1968) Snap freezing under high pressure: a new Dahl, R. & Staehelin, L.A. (1989) High-pressure freezing for the fixation technique for freeze-etching. Proceedings of the 4th Eur. Reg. preservation of biological structure: theory and practice. J Electron Conference Electron Microsc., Rome, Vol. 2, 33–34. Microsc Tech 13, 165–174. Muller-Reichert,¨ T., Hohenberg, H., O’Toole, E.T. & McDonald, K. (2003) Debenedetti, P.G. (2003) Supercooled and glassy water. J. Phys.: Condens. Cryoimmobilization and three-dimensional visualization of C. elegans Matter 15, 1669–1726. ultrastructure. J. Microsc. 212(Pt 1), 71–80. Dubochet, J. (1995) High-pressure freezing for cryoelectron microscopy. Muller-Reichert,¨ T., Mantler,¨ J., Srayko, M. & O’Toole, E. (2008) Electron Trends Cell Biol. 5(9), 366–368. microscopy of the early Caenorhabditis elegans embryo. J Microsc. 230(Pt Dubochet, J. & McDowall, A.W. (1981) Vitrification of pure water for 2), 297–307. electron microscopy. J. Microsc. 124, RP3–RP4. Murray, S. (2008) High pressure freezing and freeze substitution of Dumay, E., Picart, L., Regnault, S. & Thiebaud, M. (2006) High pressure- Schizosaccharomyces pombe and Saccharomyces cerevisiae for TEM. low temperature processing of food proteins. Biochim Biophys Acta Methods Cell Biol. 88, 3–17. 1764(3), 599–618. Richter, K. (1994) High-density morphologies of ice in high-pressure Eisenberg,D.&Kauzmann,W.(1969)TheStructureandPropertiesofWater. frozen biological specimens. Ultramicroscopy 53(3), 237–249. Oxford University Press, Oxford. Rubinsky, B., Perez, P.A. & Carlson, M.E. (2005) The thermodynamic Erk, I., Nicolas, G., Caroff, A. & Lepault, J. (1998) Electron microscopy principles of isochoric cryopreservation. Cryobiology 50, 121– of frozen biological objects: a study using cryosectioning and 138. cryosubstitution. J Microsc. 189(Pt 3), 236–248. Shimoni, E. & Muller,¨ M. (1998) On optimizing high-pressure freezing: Franks, F. (1977) Biological freezing and cryofixation. J. Microsc. 111, fromheattransfertheorytoanewmicrobiopsydevice.J.Microsc.192(Pt 3–16. 3), 236–247. Franks, F. (2003) Scientific and technological aspects of aqueous glasses. Studer, D., Michel, M. & Muller,¨ M. (1989) High pressure freezing comes Biophysical Chemistry 105, 251–261. of age. Scanning Microsc. Suppl. 3, 253–268. Gilkey, J.C. & Staehelin, L.A (1986) Advances in ultrarapid freezing for Studer, D., Michel, M., Wohlwend, M., Hunziker, E.B. & Buschmann, M.D. the preservation of cellular ultrastructure. J. Electron. Microsc. Tech. 3, (1995) Vitrification of articular cartilage by high-pressure freezing. J 177–210. Microsc. 179(Pt 3), 321–332. Hare, D.E. & Sorensen, C.M. (1987) The density of supercooled water. Szobota, S.A. & Rubinsky, B. (2006) Analysis of isochoric subcooling. II. Bulk samples cooled to the homogeneous nucleation limit. J. Chem. Cryobiology 53(1), 139–142. Phys. 87, 4840–4845. Tokuyasu, K.T. (1973) A technique for ultracryotomy of cell suspensions Hawes, P., Netherton, C.L., Mueller, M., Wileman, T. & Monaghan, and tissues. J. Cell Biol. 57, 551–565. P. (2007) Rapid freeze-substitution preserves membranes in high- Triffo, W.J., Palsdottir, H., McDonald, K.L., et al. (2008) Controlled pressure frozen tissue culture cells. J Microsc. 226, 182–189. microaspiration for high-pressure freezing: a new method for Hayakawa, K., Ueno, Y., Kawamura, S., Kato, T. & Hayashi, R. (1998) ultrastructural preservation of fragile and sparse tissues for TEM and Microorganism inactivation using high-pressure generation in sealed electron tomography. J Microsc. 230(Pt 2), 278–287. vessels under sub-zero temperature. Appl Microbiol Biotechnol. 50(4), van Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B.M. & Slot, J.W. 415–418. (2007) Immunogold labeling of cryosections from high-pressure frozen Hayashi, Y. & Nishimura, K. (2002) Application of high pressure induced cells. Traffic 8(5), 471–485. by water-freezing to the Michael reaction of alcohols with α,β-Enones. van Harreveld, A. & Crowell, J. (1964) Electron Microscopy after rapid Chemistry Lett. 2002, 296–297. freezing on a metal surface and substitution fixation. Anat. Rec. 149, Hayashi, Y., Okado, K., Ashimine, I. & Shoji, M. (2002) The Baylis– 381–386. Hillman reaction under high pressure induced by water-freezing. Vanhecke, D., Graber, W. & Studer, D. (2008) Close-to-native Tetrahedron Lett. 43, 8683–8686. ultrastructural preservation by high pressure freezing. Methods Cell Kanno, H. & Angell, C.A. (1979) Water: anomalous compressibilities to Biol. 88, 151–164. 1.9 kbar and correlation with supercooling limits. J. Chem. Phys. 70, Walther, P. (2003) Recent progress in freeze-fracturing of high-pressure 4008–4016. frozen samples. J. Microsc. 212(Pt 1), 34–43.

C 2009 The Authors Journal compilation C 2009 The Royal Microscopical Society, Journal of Microscopy, 235, 25–35