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Chapter 1 Advances in High-Pressure and Plunge-Freeze Fixation
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CHAPTER 1
Advances in High-pressure and Plunge-Freeze Fixation M. E. Galway,*J. W. Heckman, Jr.,t G. J. Hyde,s and L. C. Fowke§ 'Department of Biology The University of Michigan Ann Arbor, Michigan 48109-1048 +Center for Electron Optics Michigan State University East Lansing, Michigan 48824-131 1 *Department of Biology York University North York, Ontario M3J 1P3, Canada $Department of Biology University of Saskatchewan Saskatoon, Saskatchewan S7N OWO, Canada
I. Introduction A. Chemical Fixation versus Freeze Fixation B. Plunge Freezing and High-pressure Freezing C. Suitable Specimens for Freeze Fixation D. Hazards and Safety Precautions 11. Plunge-Freeze Fixation A. Materials and Method B. Critical Aspects of the Procedure 111. High-pressure Freeze Fixation A. Materials and Method B. Critical Aspects of the Procedure IV. Results and Discussion V. Conclusions and Perspectives References METHODS IN CELL BIOLOGY, VOL. 49 Copyright 0 1995 by Academic Press, Inc. All rightr of reproduction in any form reserved
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I. Introduction A. Chemical Fixation versus Freeze Fixation
One of the conundrums facing cell biologists is that many of the methods that are employed for investigating the natural structure and function of living cells result in the injury and, in many cases, death of the cells that are being examined. In structural investigations, a compromise is sought, in which “fixation” is employed to kill the cells in a manner that stabilizes and preserves them in as lifelike a state as possible for subsequent investigation. Ideally, fixation should be achieved too rapidly for cells to react to the process. The effectiveness of fixation by reactive chemicals, for example, glutaraldehyde, which fixes cells via cross-linking proteins, depends on the speed of diffusion of the fixative into the specimen and on its reactivity with the molecules that it encounters. It has been found that chemical fixation is too slow to capture rapid cellular processes such as membrane fusion (Menco, 1986, and references therein). It has further been shown that cells can undergo dramatic structural changes between the time of first contact with chemical fixatives and completion of the fixation process, and that they are subject to loss or displacement of poorly fixed and water-soluble components (reviewed by Gilkey and Staehelin, 1986;Hyde et al., 1991b; Kaminskyj et al., 1992). An alternative to chemical fixation is freeze fixation (cryofixation). The major advantage of cryofixation is that the physical structure of cells is stabilized much faster through reducing their thermal energy than through chemical cross-linking. Once frozen, further structural changes are almost completely inhibited in cells maintained below about -143°C (Bachman and Mayer, 1987). In order to prevent metabolic and structural changes from occurring in response to the falling temperature, the cooling rate during freezing should be very high. Water, the most abundant molecule in living cells, presents the major obstacle to achieving good cryofixation because the most thermodynamically stable state for frozen water is crystalline. Ice crystal growth during or after the freezing process severely disrupts cytoplasmic structure by displacing solutes and other cytoplasmic contents to the edges of the crystals. When the primary concern is the preservation of cellular ultrastructure in a lifelike state, the empirical goal of cryofixation is to freeze the samples so rapidly (about lO,OOO”C/s)that growth of ice crystals is restricted to less than about 10 nm in diameter (Gilkey and Staehelin, 1986; Moor, 1987; Dahl and Staehelin, 1989). These cause no detectable displacement or distortion of cellular contents at magnifications normally used for biological specimens in transmission electron microscopy. (TEM; magnifications are 20,000-40,000X). Once successfully frozen, cells and tissues can be visualized and analyzed by a variety of techniques, including low-temperature scanning electron microscopy and low temperature TEM of ultrathin cryosections. Replicas can be made of freeze-fractured or freeze-etched specimens for study at normal temperature by
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electron microscopy, or the specimens can themselves be examined at room temperature after freeze drying or' freeze substitution. These methods can be combined with various analytical methods, such as electron probe microanalysis, cytochemistryhmmunocytochemistry, and autoradiography (reviewed by Plattner and Bachman, 1982; Menco, 1986; Steinbrecht and Zierold, 1987; Robards, 1991). Recent advances in cryofixation have focused on improving the methods of heat removal from specimens, thereby increasing the size of specimens that can be successfully frozen. H u e we describe the application of two rapid freezing methods for the successful preservation of plant cell ultrastructure. One of these, plunge freezing, is inexpensive and simple, but only in the outer 10-20-pm layer of a specimen will freezing be rapid enough to ensure good preservation. The other, high-pressure (hyperbaric) freezing, requires a specially designed apparatus, but is theoretically capable of preserving specimens that are 500-600 pm thick (Gilkey and Staehelin, 1986; Moor, 1987). Detailed discussions of the theory and techniques of freeze fixation can be found in Robards and Sleytr (1985), Gilkey and Staehelin (1986), Steinbrecht and Zierold (1987), Studer et al. (1989), Roos and Morgan (1990), and Robards (1991). B. Plunge Freezing and High-pressure Freezing
In plunge freezing, liquid nitrogen at its boiling point (- 196°C) is used to cool a secondary cryogenic liquid, such as propane (which is liquid between -42 and -187°C). Specimens are frozen by plunging them rapidly and deeply into the secondary cryogen: the flow of cryogen over specimen surfaces ensures rapid dissipation of heat. The low boiling point of liquid nitrogen prevents it from being used directly on specimens in plunge freezing, since it boils on contact with the warm specimen. The resultant insulating layer of gas slows freezing sufficiently to allow large and disruptive ice crystals to form in cells (Sitte et al., 1987). Even adiabatically frozen nitrogen slush (about -209°C) is unsuitable for any but the smallest specimens due to the limited heat capacity of the medium. Pressurization of liquid nitrogen inhibits boiling, allowing it to be used directly on specimens for high-pressure freezing (Moor, 1987). The concept and method of freezing biological specimens under high pressure were developed by Riehle, Moor, and their colleagues from the late 1960s to the 1980s and resulted in the design and manufacture of the Balzers HPM 010 high-pressure freezing machine (Moor, 1987). The technique and apparatus of high-pressure freezing continue to be refined, however, in response to new applications and results (Dahl and Staehelin, 1989; Studer et al., 1989; Sartori et al., 1993). Freezing specimens under high pressure protects them from ice crystal damage by lowering the freezing point of water and reducing the rate of ice crystal nucleation and growth (Moor, 1987; Dahl and Staehelin, 1989). The design and function of the HPM 010 is described by Moor (1987, and references therein). In brief, specimens are sandwiched between two small metal cups or plates, locked into a special holder
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which is inserted into the pressure chamber in the HPM 010 and then frozen in a 0.5-s burst of liquid nitrogen pressurized to 2100 atm. Rapid transfer of the frozen specimens to liquid nitrogen at normal pressure prevents rewarming after freezing. To prevent specimens from freezing before they are fully pressurized, warm isopropyl alcohol is injected into the chamber just ahead of the nitrogen burst. A temperature-controlled closed circuit water supply keeps the pressure chamber and alcohol supply from cooling during operation of the HPM 010. C. Suitable Specimens for Freeze Fixation
In all rapid freezing methods, the quality of freeze fixation is affected by the water and solute content of the specimen. Formation of large damaging ice crystals is reduced in specimens with naturally low water content such as seeds or seed embryos. Naturally high solute levels in the cytoplasm of some cells like phloem sieve elements depresses the freezing point and reduces ice crystal formation. Similar cryoprotection can be obtained by deliberately adding such substances to specimens before freezing. Unfortunately many cryoprotectants (e.g., glycerol) can penetrate cells and perturb cellular structure and function, contrary to the goal of preserving cellular structure in its natural state (Plattner and Bachman, 1982; Gilkey and Staehelin, 1986). Some apparently inert and nonpenetrating cryoprotectants such as dextran, polyvinylpyrrolidone, and the paraffin oil 1-hexadecene, have been used in high pressure freezing to further reduce intracellular ice crystal formation by inhibiting the formation of extracellular ice crystals (Dahl and Staehelin, 1989; Studer et al., 1989; Kiss et al., 1990). However, these cryoprotectants have been rarely used for plunge freezing (Robards, 1991). One should check that cryoprotectants have no detectable effect on specimens of interest before routine use. Since the content of free water is higher in vacuoles than in cytoplasm, highly cytoplasmic cells are, in general, better preserved by freezing than are vacuolated cells. Specimens should be as small and thin as possible, not exceeding 1 mm3, so that they have a relatively large surface area (compared to volume) to contact the cryogen and dissipate heat. Examples of suitably shaped specimens are single cells (e.g., algae, pollen tubes), long thin organs like roots and fungal hyphae, excised pieces of thin tissues such as leaves, and cells projecting from the surface of organs such as hairs. D. Hazards and Safety Precautions It is important to understand the hazards of rapid freezing methods and to know what safety precautions are necessary before attempting freeze fixation (see Robards and Sleytr, 1985; Howard and O’Donnell, 1987; Sitte et al., 1987; Roos and Morgan, 1990). Hazards include ( a ) freezing injuries due to splashed cryogens, (b) oxygen deprivation due to displacement of air by nitrogen gas from boiling liquid nitrogen, (c) explosionhre hazard due to propane gas, or to
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condensation of oxygen from the air onto the surface of liquid propane, (d ) breakage of brittle glasdplastic containers exposed to cryogenic liquids, and ( e ) pressure bursting of containers in which liquid cryogens have vaporized (e.g., liquid nitrogen may be inadvertently introduced into nitrogen-cooled specimen vials during plunge freezing experiments).
11. Plunge-Freeze Fixation A. Materials and Method
1. Materials Propane. Domestic propane gas contains sufficient adulterants to lower its freezing point to about -192°C. This feature along with the presence of olfactory indicators and low cost make it the medium of choice for routine use (Howard and O’Donnell, 1987; Ridge, 1988). Bulk propane from LP gas suppliers or propane in disposable 400-g cylinders works equally well. Liquid nitrogen. Two to three liters of liquid nitrogen stored in a cryogenic Dewar flask should be sufficient for each freezing experiment. Spark-free fume hood/cupboard in which to carry out plunge freezing, in order to prevent the accumulation of flammable heavy propane vapors. Propane reservoir and Dewar flask. The reservoir consists of a 40-ml thimble, 50 mm deep, bored into the end of an aluminum rod, which can be placed in a Dewar flask for cooling in liquid nitrogen (Fig. 1). A tight-fitting aluminum cover is placed on the thimble when it is not in use to prevent condensation of atmospheric oxygen onto the propane. Cryogenic vials and Styrofoam container. Roots are freeze-substituted in small screw-capped centrifuge vials after plunge freezing. These can be weighted on the bottoms by fitting them with an ordinary 7116-in. hex nut, and cooled in a suitably sized Styrofoam container of liquid nitrogen before use. Two or more pairs of fine forceps for plunging/manipulating roots during freezing. Heavy metal rod for melting frozen propane. Five- to six-day-old seedlings of Arabidopsis thaliana prepared according to Schiefelbein and Somerville (1990). In brief, surface-sterilized seeds are germinated and grown vertically for 5-6 days on the surface of agarose-solidified nutrient medium in sealed Petri plates under artificial illumination.
2. Method The plunge freezing equipment is assembled inside a spark-free fume hood. The aluminum rod is immersed in a Dewar of liquid nitrogen and allowed to cool until bubbling of the nitrogen ceases. The surface of the nitrogen in the Dewar should be kept up to the level of the reservoir in the end of the rod. To
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Fig. 1 Apparatus for plunge-freeze fixation. (A) Aluminum rod with reservoir bored in end at
top for holding liquid propane; tight-fitting lid for excluding air from propane is shown at left beside rod. (B) Propane is liquified in the reservoir of the aluminum rod as it sits inside an ordinary widemouthed vacuum bottle liner filled with liquid nitrogen. (C) Cryogenic vial for specimen storage and freeze substitution is a modified microcentrifuge tube. Venting holes in cap prevent pressure build-up within from vaporized cryogens and allow forceps to be inserted and used as wrench for opening cold vial. The nut at base provides stability and holds vial upright in liquid nitrogen. (D) Plunge freezing of excised seedling roots. Specimens are quickly plunged straight down into the melting propane, held for 5-10 s, then rapidly transferred to precooled cryogenic vials for freeze substitution.
liquefy the propane, the tank valve is slightly opened and propane is gently introduced at a low flow rate so that it condenses on the cold walls of the reservoir. Once the thimble is full, the top is capped with a tight-fitting aluminum
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cover and the propane is cooled until frozen. Before use, the cover is removed and most of the frozen propane is melted with a heavy metal rod, and the liquid is stirred to achieve a uniform temperature (approximately -190°C) in the reservoir. While the propane is cooling, the weighted cryogenic vials are prepared for freeze substitution by adding to each 1 ml of 1%osmium tetroxide in acetone at -80°C. This solution is solidified by standing the vials upright (but not submerged) in a Styrofoam box containing liquid nitrogen. To initiate freezing, seedlings are rapidly removed from Petri plates using one pair of fine forceps and, after pinching off and discarding the cotyledons with a second pair of forceps, they are plunged root-first rapidly and deeply into the liquid propane. After holding each root in the propane for at least 5-10 s, to ensure it reaches liquid propane temperature, it is transferred as rapidly as possible onto the frozen surface of the osmium tetroxide/acetone mixture in an already opened cryogenic vial held at liquid nitrogen temperature in the styrofoam box. The vials are transferred in the Styrofoam box into a -80°C freezer, and freeze-substitution is continued for 3 days. The samples are then rewarmed over an 8- to 12-h period in an insulated box. When the samples reach O"C, they are washed three times in anhydrous acetone to remove unreacted osmium tetroxide, warmed to room temperature, infiltrated in a graded resin series, and embedded in Spurr's resin or a Quetol-based resin formulation (Kushida, 1974) for sectioning and TEM (see also Chapter 5 , this volume). B. Critical Aspects of the Procedure
The simple manual method of plunge freezing described here is subject to variability in a number of experimental conditions (e.g., speed and angle of plunge, exact temperature of propane) which are controlled in more sophisticated plunge-freeze devices (see Robards and Sleytr, 1985; Gilkey and Staehelin, 1986; Menco, 1986; Sitte et al., 1987; Robards, 1991; and references therein). With practice, however, this method can yield good freezing of appropriate specimens (Fig. 2). Howard and O'Donnell (1987) and Ridge (1990) provide helpful illustrated descriptions of two other simple setups for manual plunge freezing of fungal and plant tissue in liquid nitrogen-cooled propane.
1. Specimen Preparation Arabidopsis seedling roots are suitable specimens because they can be grasped easily in forceps and the cylindrical roots and root hairs provide large surface areas to contact the cold propane. Specimens should be surrounded by as little external liquid as possible when plunge frozen, since water is a poor thermal conductor and will slow freezing both by insulating the specimens from direct contact with the cryogen and by releasing heat during crystallization (Gilkey
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Fig. 2 Thin section from well-preserved root hair of short-haired mutant (rhd 3) of Arabidopsis thaliana depicts a Golgi body (G) with associatedvesicles,mitochondrion (M), and rough endoplasmic reticulum (arrows). The cell wall (CW) is only lightly stained. The seedling root was plunge frozen in liquid propane and freeze substituted in 1%osmium tetroxide in acetone and embedded in Quetolbased resin mixture. Uranyl acetate and lead citrate staining. Bar, 200nm.
and Staehelin, 1986). It is preferable that some of the specimens be lost to desiccation, rather than that most be unusable due to being frozen under conditions that are too wet (Ridge, 1990). Growth of Arubidopsis seedlingson solidified agarose reduces the surface moisture of the roots. However, the root hairs will desiccate and collapse on removal from the high humidity of the Petri plates if not frozen with utmost speed.
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2. Specimen Supports The roots are held in very fine forceps of low thermal conductivity. This prevents heating the propane, which would retard specimen cooling (Robards and Sleytr 1985). Specimens that cannot be grasped directly in forceps require some form of support. Small Formvar-coated loops of fine wire seem to be the most successful design (Howard and O’Donnell, 1987; Ridge, 1990; Hyde et al., 1991b, and references therein). To avoid inadvertantly wetting and/or precooling specimens, it is essential that forceps and loops be warm and dry before each use.
3. Plunge Freezing Technique To be effective, manual plunge freezing requires constant monitoring of conditions. In order to prevent premature cooling of specimens in the cold vapor above the propane, insulated covers should be kept over the liquid nitrogen and propane, and the propane reservoir should be filled to the brim at all times. Best results are obtained when propane is maintained at a uniform temperature just above freezing point. This requires that (a) the surrounding bath of liquid nitrogen remain full, ( b ) the propane be allowed to recool after each addition of fresh propane and after freezing each specimen, (c) the propane be stirred frequently to abolish temperature gradients, and ( d ) if the propane begins to freeze, it be melted using a metal rod. For best results, plunging specimens and transferring them to the cryogenic vials requires practice. Plunging should be a sudden, brisk movement that brings the sample into the cryogen as rapidly as possible. A deep propane reservoir helps, first, because it permits a longer flow of fresh cryogen over the specimen surface during the plunge, and, second, because the operator is not inhibited by fear of the specimen hitting the bottom of the reservoir.
111. High-pressure Freeze Fixation A. Materials and Method
1. Materials
Access to HPM 010 High Pressure Freezing Machine (Bal-Tec Products, Inc., Middlebury, CT,Balzers Union, Liechtenstein): eight of these are located in the United States. Tank of liquid nitrogen, to supply HPM 010. Isopropyl alcohol. Wide-mouthed, shallow container of liquid nitrogen, in which to immerse the sample holder and remove frozen protoplasts after freezing (a Styrofoam box is suitable).
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Specimen cups forming a cavity 0.6 mm thick and 1 mm in diameter when sandwiched together (Craig et af., 1987; Dahl and Staehelin, 1989). Vegetable lecithin and chloroform, for coating specimen cups. Silicone lubricant for O-ring on specimen holder. Cryogenic vials for storage or freeze substitution of specimens after freezing. Suspension of Picea glauca protoplasts in buffered osmoticum. Ultra-low gelling temperature agarose (Sigma Type IX, Sigma Chemical Co., St. Louis, MO).
2. Method Before starting to fill the 7-liter liquid nitrogen Dewar in the HPO 010, the supply of liquid nitrogen is checked to ensure it is sufficient for operating the machine, which has a consumption rate of 10-20 liters/h (Bal-Tec specifications for the HPM 010). The isopropyl alcohol reservoir in the HPM 010 is refilled, and the water level in the closed circulation water supply checked before switching it on. After startup on the automatic setting, the machine requires about 15 min to cool down with liquid nitrogen before use. Three preliminary test freezing runs are performed using a dummy sample holder with built-in temperature sensor (HPM 010 accessory). To ensure complete freezing of protoplasts during each burst of pressurized nitrogen, the monitoring system should show that pressure is maintained for 0.45-0.5 s, with a cooling time from 0 to -50°C of 10 ms. Temperature maintenance, which is an indicator of the time available to transfer the frozen protoplasts to liquid nitrogen before thawing, should be 5-9 s. Protoplasts are isolated by enzymatic digestion of suspension-cultured white spruce embryos and resuspended in a buffered salt solution (Galway et al., 1993) that includes 0.44 M sorbitol (as an osmoticum) and 0.5% ultra-low gelling temperature agarose (Sigma Type IX, Sigma Chemical Co.). The agarose holds protoplasts together during freeze substitution following freezing. Upper specimen support cups (designed by Craig et al., 1987) are dipped in a freshly prepared solution of 100 mg/ml vegetable lecithin in chloroform and air-dried before use on a lint-free tissue. A drop of concentrated, suspended protoplasts is added to a lower specimen cup positioned in the fixed part of the unfolded hinged arm of the sample holder. The lecithin-coated upper cup is positioned on top of the lower cup, and the hinged arm is closed over the specimen cup “sandwich” and locked by rotation into the body of the handle. Protoplasts are then frozen immediately by inserting the sample holder into the pressure chamber of the HPM 010, locking it with the safety bolt and firing the nitrogen jet. The sample holder is then quickly removed and plunged into an adjacent liquid nitrogen container to prevent rewarming after freezing. The lecithin-coated upper specimen cup may separate from the specimen and the lower cup when the hinged arm is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or the sliding wedge device of Craig et af. (1987). The frozen tissue is then transferred to a vial for freeze substitution over 3 days at -79°C in 2%
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osmium tetroxide in acetone, then rewarmed and embedded in Spurr’s resin (see Galway et aL, 1993). The HPM 010 is allowed 5 min to reset before freezing another sample. Fresh silicone grease is applied to the O-ring of the specimen holder after every five freezing runs. B. Critical Aspects of the Procedure
Specimen Preparation Unlike manual plunge freezing, high-pressure freezing conditions are controlled automatically by the HPM 010. However, the preparation and handling of specimens can greatly influence the effectiveness of high-pressure freezing. Ideally, specimens should snugly fit the specimen cups to ensure the best thermal contact with the enclosing metal cups and to avoid damage (Kaeser et al., 1989). A variety of specimen cups have been described for different types of specimens (Craig et al., 1987; Moor, 1987; Welter et al., 1988; Dahl and Staehelin, 1989; Studer et al., 1989). The P. glauca protoplasts were frozen in interlocking specimen cups designed to prevent specimens from being blown out during pressurization (Craig et al., 1987). Soft tissues and cell suspensions in particular may be blown out of noninterlocking cups (Craig et al., 1987). If tissue must be excised or trimmed to fit the specimen cups, this is done just before freezing to minimize changes in cellular structure and function; the possibility of preparation-induced artifacts should be kept in mind. In contrast to plunge freezing, specimens for high-pressure freezing are normally frozen in liquid, since the remaining space around the specimen in the cups is filled with a suitable air bubble-free fluid. This provides better thermal conductivity than air alone (Dahl and Staehelin, 1989; Kiss et al., 1990), and if the viscosity of the fluid can be increased without affecting the specimens (for example, by adding agarose or a viscous cryoprotectant such as dextran: see below) it will help protect the specimens against physical damage from pressureinduced shearing or shock waves (Kiss et al., 1990). Specimens containing air pockets such as leaves are vacuum-infiltrated to avoid deformation of tissue by the collapse of adjacent air pockets under pressurization (Welter et al., 1988; Studer et al., 1989).
2. Cryoprotectants Despite the cryoprotective effects of high-pressure during freezing, many specimens frozen solely in distilled water or buffer will be severely damaged by ice crystal formation (Welter et al., 1988; Studer et al., 1989; Kiss et al., 1990). Although information on the effectiveness of different nonpenetrating cryoprotectants is still limited, addition of 15% aqueous dextran (Kiss et al., 1990) or replacement of water with l-hexadecene (Studer et al., 1989) can dramatically improve yields of well-preserved plant tissue.
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IV.Results and Discussion In root hairs, growth and cell expansion are restricted to the extreme tips of the cells. Unfortunately, these vacuolated cells are very sensitive to osmotic changes and, like many tip-growing cells, they are usually poorly preserved by chemical fixation (Ridge, 1988 Kaminskyj et al., 1992). Seedling roots of A. thaliana (Columbia wild-type) average 110 pm in diameter. Root hairs are only about 20 pm in diameter at their widest, which is small enough for many to be well-preserved throughout by plunge freezing (Fig. 2), except at the highly vacuolated hair-root junction. Root hairs that are short (C0.5 mm long), either from young wild-type or mutant plants (Schiefelbein and Somerville, 1990), are better preserved than longer hairs that become bent against the roots during plunge freezing. The quality of cytoplasmic preservation in root epidermal cells varies between cells and between different roots but, in vacuolated mature cells, at best only a thin peripheral layer of cytoplasm is well preserved. Some phloem parenchyma and phloem companion cells were well preserved, probably due to the cryoprotective effect of concentrated solutes in phloem sap. Attempts to plunge freeze protoplasts attached to various specimen supports including Formvar-coated wire loops resulted in severe ice damage. On the other hand, high-pressure freezing allowed us to recover a small proportion of wellpreserved protoplasts (Fig. 3; see also Studer et aL, 1989; Galway et al., 1993). It may be possible to increase the proportion of well-preserved protoplasts by adding dextran or other nonpenetrating cryoprotectants. Note that specimens frozen under high pressure may be more liable to ice damage caused by recrystallization during freeze substitution (Dahl and Staehelin, 1989). Although prolonged exposure to 2100 atm is lethal to living cells (Gilkey and Staehelin, 1986; Moor, 1987), cells frozen under high pressure have only 20-30 ms to react to high pressure before freezing occurs. Nevertheless, some known or suspected artifacts of high-pressure freezing have been identified in plant and fungal cells (Hyde et al., 1991a; Galway et al., 1993; and references therein). Severe ice crystal damage causes unmistakable disruption of cell structure (e.g., Fig. 2G, Gilkey and Staehelin, 1986;Figs. 3,4, Kaeser et al., 1989). Milder ice crystal damage is also easily recognized in freeze-fixed specimens at intermediate magnifications in TEM (Fig. 4). The cytoplasm appears granular due to the localized aggregation of cytoplasmic contents such as ribosomes that are excluded from growing ice crystals. In addition, microtubules are collapsed, so that the distinct hollow cores are no longer visible in longitudinal sections (not shown). The typical appearance of cytoplasm and organelles in thin sections of wellpreserved plunge frozen root hairs and high-pressure frozen protoplasts is depicted in Figs. 2 and 3, respectively. The plasma membrane of root hairs is smooth and closely appressed to the cell wall (Fig. 2). In both cell types organelle profiles are smooth and rounded; Golgi body cisternae are straight and well
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Fig. 3 (a) Thin section of protoplast prepared from an embryogenic suspension culture of Piceu gluucu frozen under high pressure in a Bal-Tec HPM 010, and then freeze-substituted in 2% osmium tetroxide in acetone and embedded in Spurr’s resin. Note nucleus (N), Vacuole (V), and numerous piastids and mitochondria. Lead citrate staining. Bar, 1600 nm. (b) Enlargement shows details of nucleus (N), Golgi bodies (G), and plastid (P). Bar, 800 nm.
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Fig. 4 Thin section of a wild-typeroot hair of Arabidopsis thaliana plunge frozen,freeze-substituted,
and stained as in Fig. 1. A gradient of increasing ice damage extends through this hair from top to bottom of figure. Damaged cytoplasm at bottom appears granular and darkly stained due to the aggregation of cytoplasm into pockets between ice crystals. Note mitochondria (M). Bar, 200 nm.
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defined (Figs. 2 and 3). Cytoplasmic ribosomes are uniformly distributed except when they are aligned along the surface of cross-sectioned endoplasmic reticulum. Contrast of endoplasmic reticulum and other cellular membranes was usually poor in stained sections of both root hairs and protoplasts. Membrane contrast, as well as the degree of extraction of cell contents, varies according to the freeze substitution and embedding protocol used (Howard and O’Donnell, 1987; Ridge, 1988, 1990; Kiss et al., 1990).
V. Conclusions and Perspectives Successful manual plunge freezing of suitable plant specimens, such as Arubidopsis root hairs, shows that rapid freezing does not require elaborate or expensive equipment. Other specimens that are not suitable for plunge freezing (such as protoplasts) can be conveniently freeze-fixed by high-pressure freezing in the Bal-Tec HPM 010. These methods undoubtedly preserved both root hairs and protoplasts in a more lifelike state than conventional chemical fixation and have been used to (a) confirm models of cellular processes derived from observation of chemically fixed cells (endocytosis in protoplasts: Galway et al., 1993) and ( b ) obtain details of cytoplasmic organization that are destroyed by conventional chemical fixation (tip growth in Arabidopsis root hairs: Galway, Heckman and Schiefelbein, unpublished results). In many other cases, the application of freeze fixation methods has significantly changed our views of plant cell function and development (Dahl and Staehelin, 1989; Studer et al., 1989; Kiss et ul., 1990; Robards, 1991; and references therein). The challenge now is to ensure that these methods come into routine use by more investigators, particularly for subcellular localization studies and for investigations of dynamic cellular processes where chemical fixation methods alone cannot be relied upon. Acknowledgments M.E.G. thanks Dr. John Schiefelbeinat the University of Michigan,Ann Arbor, for the opportunity to study Arubidopsis root hair structure and development;Drs. Margaret McCully and Martin Canny for the introduction to plunge freezing methods; and Drs. Tom Giddings and Andrew Staehelin for the introduction to high-pressure freezing. G.J.H. thanks Peter Hepler, Sue Lancelle, and Dale Callahan for his introduction to plunge and high-pressure freezing, and Martin Muller for helpful advice on freezing methods. This work was supported by grants from the National Sciences and Engineering Research Council of Canada to L.C.F. and M.E.G. (NSERC Postdoctoral Award), and from the National Science Foundation to J. Schiefelbein.
References Bachman, L., and Mayer, E. (1987). Physics of water and ice: Implications for cryofixation. In “Cryotechniques in Biological Electron Microscopy” (R. A. Steinbrecht and K. Zierold, eds.), pp. 3-34. Berlin: Springer-Verlag.
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M. E. Galway rt al. Craig, S., Gilkey, J. C., and Staehelin, L. A. (1987). Improved specimen support cups and auxiliary devices for the Balzers high pressure freezing apparatus. J. Microsc. 148, 103-106. Dahl, R., and Staehelin, L. A. (1989). High-pressure freezing for the preservation of biological structure: Theory and practice. J. Electron Microsc. Tech. W, 165-174. Galway, M. E., Rennie, P. J., and Fowke, L. C. (1993). Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozedfreeze-substituted protoplasts of white spruce (Picea glauca ). J. Cell Sci. 106, 847-858. Gilkey, J. C., and Staehelin, L. A. (1986). Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J. Electron Microsc. Tech. 3, 177-210. Howard, R. J., and O’Donnell, K. L. (1987). Freeze substitution of fungi for cytological analysis. EXP.MYCO~. 11,250-269. Hyde, G. J., Lancelle, S., Hepler, P. K., and Hardham, A. R. (1991a). Sporangial structure in Phytophthora is disrupted after high pressure freezing. Protoplasma 165,203-208. Hyde, G. J., Lancelle, S., Hepler, P. K., and Hardham, A. R. (1991b). Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes. J. Cell Sci. 100,735-746. Kaeser, W., Koyro, H.-W., and Moor, H. (1989).Cryofixation of plant tissues without pretreatment. J. Microsc. W, 279-288. Kaminskyj, S.G. W., Jackson, S. L., and Heath, I. B. (1992).Fixation induces differential polarized translocations of organelles in hyphae of Saprolegnia ferax. J. Microsc. 167,153-168. Kiss, J. Z., Giddings, Th. H., Jr., Staehelin, L. A., and Sack, F. D. (1990). Comparison of the ultrastructure of conventionally fixed and high pressure frozedfreeze substituted root tips of Nicotiana and Arabidopsis, Protoplasma 157, 64-74. Kushida, H. (1974). A new method for embedding with a low viscosity epoxy resin “Quetol 651.” J. Electron Microsc. (Jpn.) 23, 197. Menco, B. Ph. M. (1986).A survey of ultra-rapid cryofixation methods with particular emphasis on applicationsto freeze-fracturing,freeze-etching,and freeze-substitution.J. Electron Microsc. Tech. 4 177-240. Moor, H. (1987).Theory and practice of high pressure freezing. I n “Cryotechniques in Biological Electron Microscopy” (R. A. Steinbrecht and K. Zierold, eds.), pp. 175-191. Berlin: SpringerVerlag. Plattner, H., and Bachmann, L. (1982). Cryofixation: A tool in biological ultrastructural research. Int. Rev. Cytol. 79,237-304. Ridge, R. W. (1988). Freeze-substitution improves the ultrastructural preservation of legume root hairs. Bot. Mag. Tokyo 101,427-441. Ridge, R. W. (1990). A simple apparatus and technique for the rapid-freeze and freeze-substitution of single-cell algae. J. Electron Microsc. 39, 120-124. Robards, A. W.(1991). Rapid-freezing methods and their applications. I n “Electron Microscopy of Plant Cells” (J. L. Hall and C. Hawes, eds.), pp. 258-312. London: Academic Press. Robards, A. W., and Sleytr, U. B. (1985). “Low Temperature Methods in Biological Electron Microscopy.” Practical Methods in Electron Microscopy, Vol. 10 (A. M. Glauert, ed.). Amsterdam: Elsevier. Roos, N., and Morgan, A. J. (1990). “Cryopreparation of Thin Biological Specimens for Electron Microscopy: Methods and Applications.” RMS Microscopy Handbook No. 21,New York Oxford Univ. Press. Sartori, N., Richter, K., and Dubochet, J. (1993). Vitrification depth can be increased more than 10fold by high-pressure freezing. J. Microsc. 172,55-61. Schiefelbein,J. W., and Somerville,C. (1990).Genetic control of root hair development in Arabidopsis thaliana. The Plant Cell 2,235-243. Sitte, H., Edelmann, L., and Neumann, K. (1987). Cryofixation without pretreatment at ambient pressure. I n “Cryotechniques in Biological Electron Microscopy” (R. A. Steinbrecht and K. Zierold, eds.), pp. 87-113. Berlin: Springer-Verlag.
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Steinbrecht, R. A., and Zierold, K. (1987). “Cryotechniques in Biological Electron Microscopy.” Berlin: Springer-Verlag. Studer, D., Michel, M., and Milller, M. (1989). High pressure freezing comes of age. Scanning Microsc., Suppl. 3,253-269. Welter, K., Milller, M., and Mendgen, K. (1988). The hyphae of Uromyces uppendiculatus within the leaf tissue after high pressure freezing and freeze substitution. Protoplasma 147, 91-99.
CHAPTER 1
Advances in High-pressure and Plunge-Freeze Fixation M. E. Galway,*J. W. Heckman, Jr.,t G. J. Hyde,s and L. C. Fowke§ 'Department of Biology The University of Michigan Ann Arbor, Michigan 48109-1048 +Center for Electron Optics Michigan State University East Lansing, Michigan 48824-131 1 *Department of Biology York University North York, Ontario M3J 1P3, Canada $Department of Biology University of Saskatchewan Saskatoon, Saskatchewan S7N OWO, Canada
I. Introduction A. Chemical Fixation versus Freeze Fixation B. Plunge Freezing and High-pressure Freezing C. Suitable Specimens for Freeze Fixation D. Hazards and Safety Precautions 11. Plunge-Freeze Fixation A. Materials and Method B. Critical Aspects of the Procedure 111. High-pressure Freeze Fixation A. Materials and Method B. Critical Aspects of the Procedure IV. Results and Discussion V. Conclusions and Perspectives References METHODS IN CELL BIOLOGY, VOL. 49 Copyright 0 1995 by Academic Press, Inc. All rightr of reproduction in any form reserved
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I. Introduction A. Chemical Fixation versus Freeze Fixation
One of the conundrums facing cell biologists is that many of the methods that are employed for investigating the natural structure and function of living cells result in the injury and, in many cases, death of the cells that are being examined. In structural investigations, a compromise is sought, in which “fixation” is employed to kill the cells in a manner that stabilizes and preserves them in as lifelike a state as possible for subsequent investigation. Ideally, fixation should be achieved too rapidly for cells to react to the process. The effectiveness of fixation by reactive chemicals, for example, glutaraldehyde, which fixes cells via cross-linking proteins, depends on the speed of diffusion of the fixative into the specimen and on its reactivity with the molecules that it encounters. It has been found that chemical fixation is too slow to capture rapid cellular processes such as membrane fusion (Menco, 1986, and references therein). It has further been shown that cells can undergo dramatic structural changes between the time of first contact with chemical fixatives and completion of the fixation process, and that they are subject to loss or displacement of poorly fixed and water-soluble components (reviewed by Gilkey and Staehelin, 1986;Hyde et al., 1991b; Kaminskyj et al., 1992). An alternative to chemical fixation is freeze fixation (cryofixation). The major advantage of cryofixation is that the physical structure of cells is stabilized much faster through reducing their thermal energy than through chemical cross-linking. Once frozen, further structural changes are almost completely inhibited in cells maintained below about -143°C (Bachman and Mayer, 1987). In order to prevent metabolic and structural changes from occurring in response to the falling temperature, the cooling rate during freezing should be very high. Water, the most abundant molecule in living cells, presents the major obstacle to achieving good cryofixation because the most thermodynamically stable state for frozen water is crystalline. Ice crystal growth during or after the freezing process severely disrupts cytoplasmic structure by displacing solutes and other cytoplasmic contents to the edges of the crystals. When the primary concern is the preservation of cellular ultrastructure in a lifelike state, the empirical goal of cryofixation is to freeze the samples so rapidly (about lO,OOO”C/s)that growth of ice crystals is restricted to less than about 10 nm in diameter (Gilkey and Staehelin, 1986; Moor, 1987; Dahl and Staehelin, 1989). These cause no detectable displacement or distortion of cellular contents at magnifications normally used for biological specimens in transmission electron microscopy. (TEM; magnifications are 20,000-40,000X). Once successfully frozen, cells and tissues can be visualized and analyzed by a variety of techniques, including low-temperature scanning electron microscopy and low temperature TEM of ultrathin cryosections. Replicas can be made of freeze-fractured or freeze-etched specimens for study at normal temperature by
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electron microscopy, or the specimens can themselves be examined at room temperature after freeze drying or' freeze substitution. These methods can be combined with various analytical methods, such as electron probe microanalysis, cytochemistryhmmunocytochemistry, and autoradiography (reviewed by Plattner and Bachman, 1982; Menco, 1986; Steinbrecht and Zierold, 1987; Robards, 1991). Recent advances in cryofixation have focused on improving the methods of heat removal from specimens, thereby increasing the size of specimens that can be successfully frozen. H u e we describe the application of two rapid freezing methods for the successful preservation of plant cell ultrastructure. One of these, plunge freezing, is inexpensive and simple, but only in the outer 10-20-pm layer of a specimen will freezing be rapid enough to ensure good preservation. The other, high-pressure (hyperbaric) freezing, requires a specially designed apparatus, but is theoretically capable of preserving specimens that are 500-600 pm thick (Gilkey and Staehelin, 1986; Moor, 1987). Detailed discussions of the theory and techniques of freeze fixation can be found in Robards and Sleytr (1985), Gilkey and Staehelin (1986), Steinbrecht and Zierold (1987), Studer et al. (1989), Roos and Morgan (1990), and Robards (1991). B. Plunge Freezing and High-pressure Freezing
In plunge freezing, liquid nitrogen at its boiling point (- 196°C) is used to cool a secondary cryogenic liquid, such as propane (which is liquid between -42 and -187°C). Specimens are frozen by plunging them rapidly and deeply into the secondary cryogen: the flow of cryogen over specimen surfaces ensures rapid dissipation of heat. The low boiling point of liquid nitrogen prevents it from being used directly on specimens in plunge freezing, since it boils on contact with the warm specimen. The resultant insulating layer of gas slows freezing sufficiently to allow large and disruptive ice crystals to form in cells (Sitte et al., 1987). Even adiabatically frozen nitrogen slush (about -209°C) is unsuitable for any but the smallest specimens due to the limited heat capacity of the medium. Pressurization of liquid nitrogen inhibits boiling, allowing it to be used directly on specimens for high-pressure freezing (Moor, 1987). The concept and method of freezing biological specimens under high pressure were developed by Riehle, Moor, and their colleagues from the late 1960s to the 1980s and resulted in the design and manufacture of the Balzers HPM 010 high-pressure freezing machine (Moor, 1987). The technique and apparatus of high-pressure freezing continue to be refined, however, in response to new applications and results (Dahl and Staehelin, 1989; Studer et al., 1989; Sartori et al., 1993). Freezing specimens under high pressure protects them from ice crystal damage by lowering the freezing point of water and reducing the rate of ice crystal nucleation and growth (Moor, 1987; Dahl and Staehelin, 1989). The design and function of the HPM 010 is described by Moor (1987, and references therein). In brief, specimens are sandwiched between two small metal cups or plates, locked into a special holder
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which is inserted into the pressure chamber in the HPM 010 and then frozen in a 0.5-s burst of liquid nitrogen pressurized to 2100 atm. Rapid transfer of the frozen specimens to liquid nitrogen at normal pressure prevents rewarming after freezing. To prevent specimens from freezing before they are fully pressurized, warm isopropyl alcohol is injected into the chamber just ahead of the nitrogen burst. A temperature-controlled closed circuit water supply keeps the pressure chamber and alcohol supply from cooling during operation of the HPM 010. C. Suitable Specimens for Freeze Fixation
In all rapid freezing methods, the quality of freeze fixation is affected by the water and solute content of the specimen. Formation of large damaging ice crystals is reduced in specimens with naturally low water content such as seeds or seed embryos. Naturally high solute levels in the cytoplasm of some cells like phloem sieve elements depresses the freezing point and reduces ice crystal formation. Similar cryoprotection can be obtained by deliberately adding such substances to specimens before freezing. Unfortunately many cryoprotectants (e.g., glycerol) can penetrate cells and perturb cellular structure and function, contrary to the goal of preserving cellular structure in its natural state (Plattner and Bachman, 1982; Gilkey and Staehelin, 1986). Some apparently inert and nonpenetrating cryoprotectants such as dextran, polyvinylpyrrolidone, and the paraffin oil 1-hexadecene, have been used in high pressure freezing to further reduce intracellular ice crystal formation by inhibiting the formation of extracellular ice crystals (Dahl and Staehelin, 1989; Studer et al., 1989; Kiss et al., 1990). However, these cryoprotectants have been rarely used for plunge freezing (Robards, 1991). One should check that cryoprotectants have no detectable effect on specimens of interest before routine use. Since the content of free water is higher in vacuoles than in cytoplasm, highly cytoplasmic cells are, in general, better preserved by freezing than are vacuolated cells. Specimens should be as small and thin as possible, not exceeding 1 mm3, so that they have a relatively large surface area (compared to volume) to contact the cryogen and dissipate heat. Examples of suitably shaped specimens are single cells (e.g., algae, pollen tubes), long thin organs like roots and fungal hyphae, excised pieces of thin tissues such as leaves, and cells projecting from the surface of organs such as hairs. D. Hazards and Safety Precautions It is important to understand the hazards of rapid freezing methods and to know what safety precautions are necessary before attempting freeze fixation (see Robards and Sleytr, 1985; Howard and O’Donnell, 1987; Sitte et al., 1987; Roos and Morgan, 1990). Hazards include ( a ) freezing injuries due to splashed cryogens, (b) oxygen deprivation due to displacement of air by nitrogen gas from boiling liquid nitrogen, (c) explosionhre hazard due to propane gas, or to
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condensation of oxygen from the air onto the surface of liquid propane, (d ) breakage of brittle glasdplastic containers exposed to cryogenic liquids, and ( e ) pressure bursting of containers in which liquid cryogens have vaporized (e.g., liquid nitrogen may be inadvertently introduced into nitrogen-cooled specimen vials during plunge freezing experiments).
11. Plunge-Freeze Fixation A. Materials and Method
1. Materials Propane. Domestic propane gas contains sufficient adulterants to lower its freezing point to about -192°C. This feature along with the presence of olfactory indicators and low cost make it the medium of choice for routine use (Howard and O’Donnell, 1987; Ridge, 1988). Bulk propane from LP gas suppliers or propane in disposable 400-g cylinders works equally well. Liquid nitrogen. Two to three liters of liquid nitrogen stored in a cryogenic Dewar flask should be sufficient for each freezing experiment. Spark-free fume hood/cupboard in which to carry out plunge freezing, in order to prevent the accumulation of flammable heavy propane vapors. Propane reservoir and Dewar flask. The reservoir consists of a 40-ml thimble, 50 mm deep, bored into the end of an aluminum rod, which can be placed in a Dewar flask for cooling in liquid nitrogen (Fig. 1). A tight-fitting aluminum cover is placed on the thimble when it is not in use to prevent condensation of atmospheric oxygen onto the propane. Cryogenic vials and Styrofoam container. Roots are freeze-substituted in small screw-capped centrifuge vials after plunge freezing. These can be weighted on the bottoms by fitting them with an ordinary 7116-in. hex nut, and cooled in a suitably sized Styrofoam container of liquid nitrogen before use. Two or more pairs of fine forceps for plunging/manipulating roots during freezing. Heavy metal rod for melting frozen propane. Five- to six-day-old seedlings of Arabidopsis thaliana prepared according to Schiefelbein and Somerville (1990). In brief, surface-sterilized seeds are germinated and grown vertically for 5-6 days on the surface of agarose-solidified nutrient medium in sealed Petri plates under artificial illumination.
2. Method The plunge freezing equipment is assembled inside a spark-free fume hood. The aluminum rod is immersed in a Dewar of liquid nitrogen and allowed to cool until bubbling of the nitrogen ceases. The surface of the nitrogen in the Dewar should be kept up to the level of the reservoir in the end of the rod. To
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Fig. 1 Apparatus for plunge-freeze fixation. (A) Aluminum rod with reservoir bored in end at
top for holding liquid propane; tight-fitting lid for excluding air from propane is shown at left beside rod. (B) Propane is liquified in the reservoir of the aluminum rod as it sits inside an ordinary widemouthed vacuum bottle liner filled with liquid nitrogen. (C) Cryogenic vial for specimen storage and freeze substitution is a modified microcentrifuge tube. Venting holes in cap prevent pressure build-up within from vaporized cryogens and allow forceps to be inserted and used as wrench for opening cold vial. The nut at base provides stability and holds vial upright in liquid nitrogen. (D) Plunge freezing of excised seedling roots. Specimens are quickly plunged straight down into the melting propane, held for 5-10 s, then rapidly transferred to precooled cryogenic vials for freeze substitution.
liquefy the propane, the tank valve is slightly opened and propane is gently introduced at a low flow rate so that it condenses on the cold walls of the reservoir. Once the thimble is full, the top is capped with a tight-fitting aluminum
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cover and the propane is cooled until frozen. Before use, the cover is removed and most of the frozen propane is melted with a heavy metal rod, and the liquid is stirred to achieve a uniform temperature (approximately -190°C) in the reservoir. While the propane is cooling, the weighted cryogenic vials are prepared for freeze substitution by adding to each 1 ml of 1%osmium tetroxide in acetone at -80°C. This solution is solidified by standing the vials upright (but not submerged) in a Styrofoam box containing liquid nitrogen. To initiate freezing, seedlings are rapidly removed from Petri plates using one pair of fine forceps and, after pinching off and discarding the cotyledons with a second pair of forceps, they are plunged root-first rapidly and deeply into the liquid propane. After holding each root in the propane for at least 5-10 s, to ensure it reaches liquid propane temperature, it is transferred as rapidly as possible onto the frozen surface of the osmium tetroxide/acetone mixture in an already opened cryogenic vial held at liquid nitrogen temperature in the styrofoam box. The vials are transferred in the Styrofoam box into a -80°C freezer, and freeze-substitution is continued for 3 days. The samples are then rewarmed over an 8- to 12-h period in an insulated box. When the samples reach O"C, they are washed three times in anhydrous acetone to remove unreacted osmium tetroxide, warmed to room temperature, infiltrated in a graded resin series, and embedded in Spurr's resin or a Quetol-based resin formulation (Kushida, 1974) for sectioning and TEM (see also Chapter 5 , this volume). B. Critical Aspects of the Procedure
The simple manual method of plunge freezing described here is subject to variability in a number of experimental conditions (e.g., speed and angle of plunge, exact temperature of propane) which are controlled in more sophisticated plunge-freeze devices (see Robards and Sleytr, 1985; Gilkey and Staehelin, 1986; Menco, 1986; Sitte et al., 1987; Robards, 1991; and references therein). With practice, however, this method can yield good freezing of appropriate specimens (Fig. 2). Howard and O'Donnell (1987) and Ridge (1990) provide helpful illustrated descriptions of two other simple setups for manual plunge freezing of fungal and plant tissue in liquid nitrogen-cooled propane.
1. Specimen Preparation Arabidopsis seedling roots are suitable specimens because they can be grasped easily in forceps and the cylindrical roots and root hairs provide large surface areas to contact the cold propane. Specimens should be surrounded by as little external liquid as possible when plunge frozen, since water is a poor thermal conductor and will slow freezing both by insulating the specimens from direct contact with the cryogen and by releasing heat during crystallization (Gilkey
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Fig. 2 Thin section from well-preserved root hair of short-haired mutant (rhd 3) of Arabidopsis thaliana depicts a Golgi body (G) with associatedvesicles,mitochondrion (M), and rough endoplasmic reticulum (arrows). The cell wall (CW) is only lightly stained. The seedling root was plunge frozen in liquid propane and freeze substituted in 1%osmium tetroxide in acetone and embedded in Quetolbased resin mixture. Uranyl acetate and lead citrate staining. Bar, 200nm.
and Staehelin, 1986). It is preferable that some of the specimens be lost to desiccation, rather than that most be unusable due to being frozen under conditions that are too wet (Ridge, 1990). Growth of Arubidopsis seedlingson solidified agarose reduces the surface moisture of the roots. However, the root hairs will desiccate and collapse on removal from the high humidity of the Petri plates if not frozen with utmost speed.
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2. Specimen Supports The roots are held in very fine forceps of low thermal conductivity. This prevents heating the propane, which would retard specimen cooling (Robards and Sleytr 1985). Specimens that cannot be grasped directly in forceps require some form of support. Small Formvar-coated loops of fine wire seem to be the most successful design (Howard and O’Donnell, 1987; Ridge, 1990; Hyde et al., 1991b, and references therein). To avoid inadvertantly wetting and/or precooling specimens, it is essential that forceps and loops be warm and dry before each use.
3. Plunge Freezing Technique To be effective, manual plunge freezing requires constant monitoring of conditions. In order to prevent premature cooling of specimens in the cold vapor above the propane, insulated covers should be kept over the liquid nitrogen and propane, and the propane reservoir should be filled to the brim at all times. Best results are obtained when propane is maintained at a uniform temperature just above freezing point. This requires that (a) the surrounding bath of liquid nitrogen remain full, ( b ) the propane be allowed to recool after each addition of fresh propane and after freezing each specimen, (c) the propane be stirred frequently to abolish temperature gradients, and ( d ) if the propane begins to freeze, it be melted using a metal rod. For best results, plunging specimens and transferring them to the cryogenic vials requires practice. Plunging should be a sudden, brisk movement that brings the sample into the cryogen as rapidly as possible. A deep propane reservoir helps, first, because it permits a longer flow of fresh cryogen over the specimen surface during the plunge, and, second, because the operator is not inhibited by fear of the specimen hitting the bottom of the reservoir.
111. High-pressure Freeze Fixation A. Materials and Method
1. Materials
Access to HPM 010 High Pressure Freezing Machine (Bal-Tec Products, Inc., Middlebury, CT,Balzers Union, Liechtenstein): eight of these are located in the United States. Tank of liquid nitrogen, to supply HPM 010. Isopropyl alcohol. Wide-mouthed, shallow container of liquid nitrogen, in which to immerse the sample holder and remove frozen protoplasts after freezing (a Styrofoam box is suitable).
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Specimen cups forming a cavity 0.6 mm thick and 1 mm in diameter when sandwiched together (Craig et af., 1987; Dahl and Staehelin, 1989). Vegetable lecithin and chloroform, for coating specimen cups. Silicone lubricant for O-ring on specimen holder. Cryogenic vials for storage or freeze substitution of specimens after freezing. Suspension of Picea glauca protoplasts in buffered osmoticum. Ultra-low gelling temperature agarose (Sigma Type IX, Sigma Chemical Co., St. Louis, MO).
2. Method Before starting to fill the 7-liter liquid nitrogen Dewar in the HPO 010, the supply of liquid nitrogen is checked to ensure it is sufficient for operating the machine, which has a consumption rate of 10-20 liters/h (Bal-Tec specifications for the HPM 010). The isopropyl alcohol reservoir in the HPM 010 is refilled, and the water level in the closed circulation water supply checked before switching it on. After startup on the automatic setting, the machine requires about 15 min to cool down with liquid nitrogen before use. Three preliminary test freezing runs are performed using a dummy sample holder with built-in temperature sensor (HPM 010 accessory). To ensure complete freezing of protoplasts during each burst of pressurized nitrogen, the monitoring system should show that pressure is maintained for 0.45-0.5 s, with a cooling time from 0 to -50°C of 10 ms. Temperature maintenance, which is an indicator of the time available to transfer the frozen protoplasts to liquid nitrogen before thawing, should be 5-9 s. Protoplasts are isolated by enzymatic digestion of suspension-cultured white spruce embryos and resuspended in a buffered salt solution (Galway et al., 1993) that includes 0.44 M sorbitol (as an osmoticum) and 0.5% ultra-low gelling temperature agarose (Sigma Type IX, Sigma Chemical Co.). The agarose holds protoplasts together during freeze substitution following freezing. Upper specimen support cups (designed by Craig et al., 1987) are dipped in a freshly prepared solution of 100 mg/ml vegetable lecithin in chloroform and air-dried before use on a lint-free tissue. A drop of concentrated, suspended protoplasts is added to a lower specimen cup positioned in the fixed part of the unfolded hinged arm of the sample holder. The lecithin-coated upper cup is positioned on top of the lower cup, and the hinged arm is closed over the specimen cup “sandwich” and locked by rotation into the body of the handle. Protoplasts are then frozen immediately by inserting the sample holder into the pressure chamber of the HPM 010, locking it with the safety bolt and firing the nitrogen jet. The sample holder is then quickly removed and plunged into an adjacent liquid nitrogen container to prevent rewarming after freezing. The lecithin-coated upper specimen cup may separate from the specimen and the lower cup when the hinged arm is opened with forceps under liquid nitrogen; if not, the cups are separated using forceps or the sliding wedge device of Craig et af. (1987). The frozen tissue is then transferred to a vial for freeze substitution over 3 days at -79°C in 2%
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osmium tetroxide in acetone, then rewarmed and embedded in Spurr’s resin (see Galway et aL, 1993). The HPM 010 is allowed 5 min to reset before freezing another sample. Fresh silicone grease is applied to the O-ring of the specimen holder after every five freezing runs. B. Critical Aspects of the Procedure
Specimen Preparation Unlike manual plunge freezing, high-pressure freezing conditions are controlled automatically by the HPM 010. However, the preparation and handling of specimens can greatly influence the effectiveness of high-pressure freezing. Ideally, specimens should snugly fit the specimen cups to ensure the best thermal contact with the enclosing metal cups and to avoid damage (Kaeser et al., 1989). A variety of specimen cups have been described for different types of specimens (Craig et al., 1987; Moor, 1987; Welter et al., 1988; Dahl and Staehelin, 1989; Studer et al., 1989). The P. glauca protoplasts were frozen in interlocking specimen cups designed to prevent specimens from being blown out during pressurization (Craig et al., 1987). Soft tissues and cell suspensions in particular may be blown out of noninterlocking cups (Craig et al., 1987). If tissue must be excised or trimmed to fit the specimen cups, this is done just before freezing to minimize changes in cellular structure and function; the possibility of preparation-induced artifacts should be kept in mind. In contrast to plunge freezing, specimens for high-pressure freezing are normally frozen in liquid, since the remaining space around the specimen in the cups is filled with a suitable air bubble-free fluid. This provides better thermal conductivity than air alone (Dahl and Staehelin, 1989; Kiss et al., 1990), and if the viscosity of the fluid can be increased without affecting the specimens (for example, by adding agarose or a viscous cryoprotectant such as dextran: see below) it will help protect the specimens against physical damage from pressureinduced shearing or shock waves (Kiss et al., 1990). Specimens containing air pockets such as leaves are vacuum-infiltrated to avoid deformation of tissue by the collapse of adjacent air pockets under pressurization (Welter et al., 1988; Studer et al., 1989).
2. Cryoprotectants Despite the cryoprotective effects of high-pressure during freezing, many specimens frozen solely in distilled water or buffer will be severely damaged by ice crystal formation (Welter et al., 1988; Studer et al., 1989; Kiss et al., 1990). Although information on the effectiveness of different nonpenetrating cryoprotectants is still limited, addition of 15% aqueous dextran (Kiss et al., 1990) or replacement of water with l-hexadecene (Studer et al., 1989) can dramatically improve yields of well-preserved plant tissue.
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IV.Results and Discussion In root hairs, growth and cell expansion are restricted to the extreme tips of the cells. Unfortunately, these vacuolated cells are very sensitive to osmotic changes and, like many tip-growing cells, they are usually poorly preserved by chemical fixation (Ridge, 1988 Kaminskyj et al., 1992). Seedling roots of A. thaliana (Columbia wild-type) average 110 pm in diameter. Root hairs are only about 20 pm in diameter at their widest, which is small enough for many to be well-preserved throughout by plunge freezing (Fig. 2), except at the highly vacuolated hair-root junction. Root hairs that are short (C0.5 mm long), either from young wild-type or mutant plants (Schiefelbein and Somerville, 1990), are better preserved than longer hairs that become bent against the roots during plunge freezing. The quality of cytoplasmic preservation in root epidermal cells varies between cells and between different roots but, in vacuolated mature cells, at best only a thin peripheral layer of cytoplasm is well preserved. Some phloem parenchyma and phloem companion cells were well preserved, probably due to the cryoprotective effect of concentrated solutes in phloem sap. Attempts to plunge freeze protoplasts attached to various specimen supports including Formvar-coated wire loops resulted in severe ice damage. On the other hand, high-pressure freezing allowed us to recover a small proportion of wellpreserved protoplasts (Fig. 3; see also Studer et aL, 1989; Galway et al., 1993). It may be possible to increase the proportion of well-preserved protoplasts by adding dextran or other nonpenetrating cryoprotectants. Note that specimens frozen under high pressure may be more liable to ice damage caused by recrystallization during freeze substitution (Dahl and Staehelin, 1989). Although prolonged exposure to 2100 atm is lethal to living cells (Gilkey and Staehelin, 1986; Moor, 1987), cells frozen under high pressure have only 20-30 ms to react to high pressure before freezing occurs. Nevertheless, some known or suspected artifacts of high-pressure freezing have been identified in plant and fungal cells (Hyde et al., 1991a; Galway et al., 1993; and references therein). Severe ice crystal damage causes unmistakable disruption of cell structure (e.g., Fig. 2G, Gilkey and Staehelin, 1986;Figs. 3,4, Kaeser et al., 1989). Milder ice crystal damage is also easily recognized in freeze-fixed specimens at intermediate magnifications in TEM (Fig. 4). The cytoplasm appears granular due to the localized aggregation of cytoplasmic contents such as ribosomes that are excluded from growing ice crystals. In addition, microtubules are collapsed, so that the distinct hollow cores are no longer visible in longitudinal sections (not shown). The typical appearance of cytoplasm and organelles in thin sections of wellpreserved plunge frozen root hairs and high-pressure frozen protoplasts is depicted in Figs. 2 and 3, respectively. The plasma membrane of root hairs is smooth and closely appressed to the cell wall (Fig. 2). In both cell types organelle profiles are smooth and rounded; Golgi body cisternae are straight and well
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Fig. 3 (a) Thin section of protoplast prepared from an embryogenic suspension culture of Piceu gluucu frozen under high pressure in a Bal-Tec HPM 010, and then freeze-substituted in 2% osmium tetroxide in acetone and embedded in Spurr’s resin. Note nucleus (N), Vacuole (V), and numerous piastids and mitochondria. Lead citrate staining. Bar, 1600 nm. (b) Enlargement shows details of nucleus (N), Golgi bodies (G), and plastid (P). Bar, 800 nm.
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Fig. 4 Thin section of a wild-typeroot hair of Arabidopsis thaliana plunge frozen,freeze-substituted,
and stained as in Fig. 1. A gradient of increasing ice damage extends through this hair from top to bottom of figure. Damaged cytoplasm at bottom appears granular and darkly stained due to the aggregation of cytoplasm into pockets between ice crystals. Note mitochondria (M). Bar, 200 nm.
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defined (Figs. 2 and 3). Cytoplasmic ribosomes are uniformly distributed except when they are aligned along the surface of cross-sectioned endoplasmic reticulum. Contrast of endoplasmic reticulum and other cellular membranes was usually poor in stained sections of both root hairs and protoplasts. Membrane contrast, as well as the degree of extraction of cell contents, varies according to the freeze substitution and embedding protocol used (Howard and O’Donnell, 1987; Ridge, 1988, 1990; Kiss et al., 1990).
V. Conclusions and Perspectives Successful manual plunge freezing of suitable plant specimens, such as Arubidopsis root hairs, shows that rapid freezing does not require elaborate or expensive equipment. Other specimens that are not suitable for plunge freezing (such as protoplasts) can be conveniently freeze-fixed by high-pressure freezing in the Bal-Tec HPM 010. These methods undoubtedly preserved both root hairs and protoplasts in a more lifelike state than conventional chemical fixation and have been used to (a) confirm models of cellular processes derived from observation of chemically fixed cells (endocytosis in protoplasts: Galway et al., 1993) and ( b ) obtain details of cytoplasmic organization that are destroyed by conventional chemical fixation (tip growth in Arabidopsis root hairs: Galway, Heckman and Schiefelbein, unpublished results). In many other cases, the application of freeze fixation methods has significantly changed our views of plant cell function and development (Dahl and Staehelin, 1989; Studer et al., 1989; Kiss et ul., 1990; Robards, 1991; and references therein). The challenge now is to ensure that these methods come into routine use by more investigators, particularly for subcellular localization studies and for investigations of dynamic cellular processes where chemical fixation methods alone cannot be relied upon. Acknowledgments M.E.G. thanks Dr. John Schiefelbeinat the University of Michigan,Ann Arbor, for the opportunity to study Arubidopsis root hair structure and development;Drs. Margaret McCully and Martin Canny for the introduction to plunge freezing methods; and Drs. Tom Giddings and Andrew Staehelin for the introduction to high-pressure freezing. G.J.H. thanks Peter Hepler, Sue Lancelle, and Dale Callahan for his introduction to plunge and high-pressure freezing, and Martin Muller for helpful advice on freezing methods. This work was supported by grants from the National Sciences and Engineering Research Council of Canada to L.C.F. and M.E.G. (NSERC Postdoctoral Award), and from the National Science Foundation to J. Schiefelbein.
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