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 water. 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 ice 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