An Automated Device for Cryofixation of Specimens of Electron Microscopy Using Liquid Helium

Technical Advance: An Automated Device for Cryofixation of Specimens of Electron Microscopy using Liquid Helium

Akiko Hisada 1, 4,TomokoYoshida1, 2, Shigeo Kubota 1, 5,NaokoK.Nishizawa3 and Masaki Furuya 1 1 Hitachi Advanced Research Laboratory, Hatoyama, Saitama, 350-0395 Japan 2 Hitachi Instruments Service Co., Yotsuya, Shinjuku-ku, Tokyo, 160-0004 Japan 3 The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan

; Metal-contact rapid freezing using liquid helium is the- (TEM). For rapid processes, temporal resolution is also impor- oretically the best method for preserving the fine structure tant (Van Harreveld et al. 1974, Heuser et al. 1976, Heuser et of living cells with high temporal resolution in preparation al. 1979). However, TEM often fails to preserve the precise of tissue samples for electron microscopy. However, this structural information about dynamic cellular processes, method is not commonly used, because of its technical diffi- because conventional processing consists of slow processes culty and low reproducibility. We have designed and con- such as chemical fixation and dehydration at room tempera- structed an automatic device which allows simple, rapid ture. The limitations of chemical fixation can largely be over- and reproducible preparation of high-quality electron come using the cryofixation technique. The advantage of cry- microscopic specimens by the non-specialist. We assessed ofixation over conventional processing lies mainly in the the quality of cryofixation in samples prepared using this extremely rapid physical fixation of the living specimen. The device by examining the preservation of cellular ultrastruc- time required for cryofixation by vitrification (absence of crys- ture in relation to distance from the freezing block, and talline ice in a specimen) is estimated to be less than 0.1 ms, found that the region within 10 mmofthemetal-contact which is substantially less (by a factor of 104) than that plane was fixed with the highest quality. We applied this required for chemical fixation by infiltration with aldehydes or device, in combination with freeze-substitution methods heavy metal compounds (Sitte et al. 1987). and immunocytochemical techniques, to two phenomena It was for a long time difficult to achieve adequate freez- involving rapid movement of subcellular components: (1) ing of plant materials, except in the case of single-celled algae active movement of subcellular structures in the papillar (Mita et al. 1986). However, Usukura et al. (1983) described a cells of stigma and (2) light-induced rapid subcellular manually operated liquid helium-cooled cryofixation apparatus, translocation of phytochrome A. Considering the impor- and this apparatus has been subsequently used to achieve high tance of understanding subcellular processes of living cells spatial and temporal resolution in several studies of dynamic for molecular and cell biology, this device will be a useful cellular processes in plants (Shojima et al. 1987, Nishizawa tool for diverse biological applications in the near future. and Mori 1989, Nishizawa et al. 1990, Nishizawa et al. 1994, Nagatani et al. 1993) and for localization of antigens, espe- Key words: Arabidopsis — Automated metal-contact cryofixa- cially water-soluble substances, by TEM immunocytochemis- tion — Electron microscopy — Immunostaining — Liquid try (Shojima et al. 1987, Nishizawa et al. 1990, Nishizawa et helium — Pea. al. 1994). Besides, the rapid-freeze technique was also used in combination with deep-etching to examine the cell wall archi- Abbreviations: BSA, bovine serum albumin; GFP, green fluores- tecture of both cultured cells and pea epidermal cells (McCann cent protein; PBS, phosphate-buffered saline; PHYA, apoprotein of phytochrome A; TEM, transmission electron microscope. et al. 1990, Itoh and Ogawa 1993, Fujino and Itoh 1998). In addition, a method for cryofixation at high pressure (approx. 210 MPa) (Müller and Moore 1984) has been reported to be well suited for plant specimens (Kiss et al. 1990, Staehe- Introduction lin et al. 1990, Galway et al. 1993, Samuels et al. 1995, Lons- dale et al. 1999). Recently, using this technique, a novel kind of Investigation of the fine structure of living cells is impor- cell plate involved in endosperm cellularization was character- tant for understanding many intracellular processes. Adequate ized (Otegui and Staehelin 2000), and precise ultrastructural spatial resolution of structures involved in subcellular proc- information of nodal endoplasmic reticulum of columella root esses can be achieved using transmission electron microscopy cap cells was gained (Zheng and Staehelin 2001).

4 Corresponding author: E-mail, [email protected]; Fax, +81-49-296-6006. 5 Present address: Kubota Techno, 7-10-1, Musashi-dai, Hidaka, Saitama, 350-1255 Japan.

885 886 Automated cryofixation device using liquid helium

Fig. 1 Schematic drawing of the sequential operation for cryofixation. Operation of the automated device is represented in sectional diagrams, shown in sequence from the initial stage (A) to the freezing stage (B) and the final stage (C). Automatic apparatus are manipulated with air pressure which is under centralized control (l) using electromagnetic valves. Grey shading indicates the apparatus working in each diagram. (A) The outer Dewar flask (f) is filled with liquid nitrogen from the liquid nitrogen port (v). The inner Dewar flask (g) is hermetically sealed to prevent the generation of frost from moisture in the air, and is filled with liquid helium from the liquid helium port (h). In the center of the inner Dewar flask (g), the copper block (b and c) is suspended from the upper panel. The lower part of copper block is settled inside the flask as a basement block (c). The upper part (b), 30 mm in diameter and 20 mm in height, is replaceable and its surface acts as the contact plane for specimens. The block port (d) and the plunger guide pipe (e) are united in one Teflon block and this unit is manually rotated. The block port (d) is used for setting up of upper copper block (b) and has the transparent window (p) in the cap that is used to check the contact plane of the block. After this operation, the plunger guide pipe (e) replaced with the block port. The plunger (j) is anchored on the automatic injector (q). The specimen holder (a) is made of silver in order to have minimum mass for optimum thermal conductivity. A fresh specimen mounted on the specimen holder (a) is placed on the tip of plunger (j). The operation starts immediately after turning on the switch button (k) on the top panel of the controller (l). (B) The plunger (j) brings the specimen to an off-center position on the surface of the copper block (b) through the guide pipe (e). The electric heater (r) maintains the inside of the guide pipe (e) at room temperature to prevent the specimen freezing before contact with the copper block (b). The shutter (m) opens just before the specimen reaches the end of the guide pipe (e). The automatic air escape (n) releases the warm air inside the guide pipe (e) just before shutter opening. The plunger (j) keeps the specimen on the copper block for a time specified by the timer (Fig. 1A, s). (C) The plunger (j) moves up to the initial position. A storage bottle (o) filled with liquid nitrogen moves to a position directly below the plunger (arrow***). The automatic ejector (t) in the plunger (j) pushes out the specimen holder (a) into the storage bottle (o). An automatic block rotator (u) located at the connection between the copper block and the upper pane automatically turns the copper block (b and c) through one-eighth of a revolution (arrows****).

Despite the fact that the cryofixation method at atmos- helium temperature which is simple to operate and therefore of pheric pressure using liquid helium has provided excellent great potential for use by non-specialists in diverse fields of preparations, very few studies have so far dealt with material biology. from higher plants. This is mainly because methods using In this paper, we introduce this new instrument to plant apparatus with a complicated manual setup is extremely time- biologists and show how fine structure is preserved in cry- consuming and gives poor reproducibility. Considerable exper- ofixed samples. Use of this instrument will significantly reduce tise has therefore been necessary for the preparation of high- the labor time and the cost for the preparation of top-quality quality sections for TEM. To overcome these problems, we specimens for TEM. have designed an automated device for cryofixation at liquid Automated cryofixation device using liquid helium 887

Results and Discussion

Performance of the automated device Automation of the rapid freezing device using liquid helium has four main advantages over manual operation, namely (1) simple operation requiring no specific skill, (2) a high reproducibility of cryofixation, (3) reduction of time necessary for preparation of multiple samples, and (4) significant reduction in running costs. First, we can put a great emphasis on extremely easy operation of this automated cryofixation device. It is possible to carry out cryofixation of living samples without skill or practice, by simply pushing the start button of this device (Fig. 1). The most important feature of the automatic device is the rapid, automated transfer of the specimen from metal-contact at liquid helium temperature (Fig. 1B) to storage in liquid nitrogen (Fig. 1C). Second, the automatic operation enabled cryofixation to be performed on multiple samples under the same conditions. Considering that one of the most important factors to preserve a fine subcellular structure was the strength of impact at the time of metal contacting (Sitte et al. 1987, Robards 1991), this was controlled mechanically by a spring on the inside of the plunger (Fig. 1B, j) and also by adjusting the amount of using air with electromagnetic valves for the plunger injector (Fig. 1B, q). These control points allow the impact at metal-contact Fig. 2 Sectional diagram of the copper block and the block rotator. to be properly adjusted according to the properties of the sam- (A) Upper view of the contact plane. (B) Front view at the time when ples. In addition to the samples of plant material reported here, the plunger (c) pushes the specimen (b) on the copper block (a). The plunger (c) brings a sample (b) to the contact position that is offset we have also confirmed the high reproducibility of freezing 10 mm horizontally from the center of the copper block (a). The auto- quality in other biological specimens including mutants of Ara- matic block rotator (f) turns the copper block (a and d, arrows) by one- bidopsis, cultured animal cells, and mice (data not shown). In eighth after contact with a sample, so that a clear surface of the block all cases that the depth of high-quality freezing was essentially becomes available for the next specimen. Eight small circles in the cop- the same as that for the plant material described above (Fig. 3, per block (A, a) indicate the contact positions with specimens (B, b). 4). Cells from the liver and small intestine of mice were examined as relatively uniform samples to evaluate the reproducibility of cryofixation. Eight pieces of each organ were frozen specimen. We were thus able to carry out uninterrupted cry- using two copper blocks and we estimated the depth of high ofixation of eight specimens using one copper block, avoiding quality fixation from the contact plane in each specimen. As a the need for removal of frost and cleaning of the apparatus result, 100% specimens showed smooth membranes including between samples, and saving a substantial amount of time. Ini- the plasma membrane and mitochondria in the region within tial setup of the device and filling with coolant took about around 10 mm from the contact plane. 20 min, while the automated operation including metal contact- Finally, incorporation of an automatic block rotator (Fig. ing (Fig. 1B), storing the specimen in liquid nitrogen (Fig. 1C) 1C, u; Fig. 2A and 2B, f) also significantly reduces the han- and block rotation (Fig. 1C), was completed within a few s. dling time needed for sequential freezing of multiple samples, After freezing the eight specimens, it took 10 min to prepare thus reducing the cost of the experiment. In order to maintain the subsequent block and to refill the coolant, before being able good thermal conductivity of the contact plane, the surface of to resume cryofixation. the cooled block must be completely clean before each cryofix- In addition to saving time, the use of automation to pre- ation. If the block is replaced for each specimen, a large pare eight specimens on one block substantially reduced the amount of liquid helium is vaporized, resulting in low effi- volume of liquid helium required, compared to processing of ciency of coolant use and loss of time due to the need for more individual samples. Initial cooling of the device from room frequent refilling. The block rotator automatically turned the temperature to around –270°C required 6 liters of liquid nitro- copper block (Fig. 1C, b and c; Fig. 2A and 2B, a and d) by gen and 20 liters of liquid helium. Processing of each subse- one-eighth after each contact with a specimen, so that a clear quent block required an additional 2 to 3 liters of liquid helium. surface of the metal became ready immediately for the next More than hundred specimens can therefore be prepared with a 888 Automated cryofixation device using liquid helium

Fig. 3 Quality of preservation of the ultrastructure in the vicinal area to the contact plane of Arabidopsis root tip. Ultrathin sections were cut perpendicular to the contact plane with the copper block. (A) The area from the contact plane to a depth of approx. 20 mm. (B), (C), (D) and (E) show magnified images of areas indicated with rectangles in (A). CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; PM, plasma membrane; V, vacuole; G, Golgi body; CP, contact plane with the copper block. Bars in (B), (C), (D) and (E) represent 0.2 mm. Automated cryofixation device using liquid helium 889

Fig. 4 Ultra structure of a papillar cell from the stigma of an Arabidopsis flower. (A) The tip area of a papillar cell. (B) Vesicles budding from Golgi bodies. (C) Secretary vesicle (arrow) in the process of fusing to the plasma membrane and releasing its contents. (D) Double membranes of chloroplast and mitochondria. (E) and (F) Semi-serial sections of chloroplast. (G) The narrow part of a chloroplast, bounded by an electron dense layer (arrows). CW, cell wall; C, chloroplast; ER, endoplasmic reticulum; G, Golgi body; M, mitochondria; PM, plasma membrane; V, vacuole; Ves, vesicle. Bars represent 0.2 mm. 890 Automated cryofixation device using liquid helium

ples show that the highest quality preservation of ultrastructure occurred within 5 mm of the contact plane (Fig. 3A and 3B). In this region, the plasma membrane (Fig. 3B, PM) and membranes of other organelles including mitochondria (Fig. 3B, M), endoplasmic reticulum (Fig. 3B, ER), vacuoles (Fig. 3B, V) and Golgi bodies (Fig. 3B, G) were smooth. Vacuoles showed a complicated form and there were amorphous network and several electron-dense spots within the vacuoles (Fig. 3A and 3B, V). The round shaped structures were electron-dense and sur- rounded by membrane, and ribosomes were parallel to the outer side of this membrane (Fig. 3B, arrows) as also seen in the rough endoplasmic reticulum (Fig. 3B, ER). This structure possibly appeared to be spindle-shape (Fig. 3A, arrows). Sev- eral such round- or spindle-shaped structures were seen (Fig. 3A), ranging in diameter from about one-third to one micron. These structures are most likely dilated cisternae of the endoplasmic reticulum, common in members of the Brassicaceae (Gunning 1998). The cryofixation method also provided resolution within the round shaped structure. The region close to the membrane was low in electron density, whereas the central region was significantly more electron-dense (Fig. 3B, arrows) compared to the endoplasmic reticulum (Fig. 3B, ER). The zone around 10 mm from the contact plane was still well preserved (Fig. 3D), but the zone around 20 mm from the contact plane showed visible damage that indicates the formation of ice crystals (Fig. 3E). High-quality freezing beyond a depth of 15–20 mm has only rarely been reported (Robards 1991) and we therefore conclude that the performance of the automated device is comparable to manual cryofixation.

Application to dynamic subcellular processes Finally, we examined the efficiency of the automated cryofixation device for capturing rapidly-moving subcellular phenomena in plant cells, using two well-known examples. Subcellular processes in papillar cells of Brassica campestris Fig. 5 Immunolabeling of phytochrome A apo-proteins in pea hook were previously described from samples prepared using a cells irradiated with continuous red light for 30 min. (A) Wild type manual metal-contacting device at liquid helium temperature (10 nm-gold particles). (B) Wild type (5 nm-gold particles). (C) (Nishizawa et al. 1990). For the purposes of comparison, we PHYA-deficient fun1-1 mutant (10 nm-gold particles). N, nucleus; therefore examined papillar cells of the stigma from Arabidop- Cyt, cytoplasm. Arrows indicate nuclear membrane. Arrowheads indi- sis flowers. Microphotographs of the papillar cells showed cate aggregation forms of PHYA immunogold. Bars indicate 0.5 mm. clear images in which we observed ribosomes, Golgi bodies, mitochondria, chloroplasts, vesicles, and vacuoles (Fig. 4A), all with well preserved fine structure. Subcellular movements such standard 50-liter tank of liquid helium. Increase the number of as secretion, budding, and membrane fusion were also cap- specimens frozen on one copper block by eight times allowed tured (Fig. 4). The Golgi stacks contained cisternae, transfer relative cost saving per specimen. And it was possible to oper- vesicles and secretory vesicles (Fig. 4B). Vesicles close to the ate the device continuously throughout the day, which resulted plasma membrane seemed to be exocytosis (Fig. 4C). The in the most economical use of coolants. quality of these samples prepared using the new automated device was equivalent to that seen in samples prepared by the Evaluation of the quality of rapid freezing previously reported manual technique. We investigated the state of ultrastructure preservation in We also observed semi-serial thin sections of chloroplasts Arabidopsis root-tip specimens prepared using the automated in papillar cells (Fig. 4E, 4F). The chloroplast was observed as cryofixation device. Electron micrographs of the resultant sam- two individual parts (Fig. 4E), joined by a narrow connecting band (Fig. 4F). Within the band, we observed a filamentous Automated cryofixation device using liquid helium 891 structure bounded by an electron-dense layer (Fig. 4G, arrows). achieved 5,000 K s–1 at the surface of a specimen, but is not These complicated forms were interpreted to be chloroplasts more than a few hundred in the center region (Moor 1987). undergoing division (Pyke 1999) and the electron-dense struc- Because temporal resolution of cryofixation depends on the ture in the narrow part (Fig. 4G) may be equivalent to the plas- cooling rate, high pressure freezing is slower process with poor tid-dividing ring that was previously reported in single cell time resolution than the freezing at atmospheric pressure (Sitte algae prepared using the rapid-freezing technique (Mita et al. et al. 1987). This method has therefore been widely used for 1986). The occurrence of a plastid dividing ring during chloro- examination of relatively large specimens, where a high degree plast division in higher plants has been observed by conven- of temporal resolution is not required. Conversely, the liquid tional chemical fixation methods (Robertson et al. 1996, Pyke helium-cooled, metal-contact cryofixation at atmospheric pres- 1999). However, the rapid freezing technique enabled us to sure was necessarily implemented at the high cooling rates of capture the dynamic changes in the shape of the chloroplast approx. 106 Ks–1 and therefore achieves vitrification with high with a high degree of temporal resolution. temporal resolution but only in a restricted area. The choice of It has recently been shown by green fluorescent protein cryofixation method should thus take into account the distinct (GFP)-fusion (Kircher et al. 1999) and immunocytochemical advantages of these two methods. techniques (Hisada et al. 2000) that phytochrome A translo- cates quickly from the cytosol to the nucleus upon light expo- Future prospects sure. Subcellular movement of phytochrome A has been In addition to the automation of the process of metal- reported to commence within 1 min of exposure to red light contact freezing, it will potentially be useful to extend the auto- (Hisada et al. 2000). Such a movement is too rapid to be mation process to include steps before or after cryofixation. For captured by conventional chemical fixation methods. An example, an automatic apparatus on the plunger of the cryofix- additional problem is that phytochrome A is a soluble protein ation device could be added, in order to apply specific treat- (Butler et al. 1959), and its subcellular localization is therefore ments (such as chemical or light treatments) to living cells on highly sensitive to disturbance during the fixation process the specimen holder. Use of such an apparatus has been (Pratt and Coleman 1974). We therefore used the rapid-freezing reported in the time-resolved analysis of interaction between technique to prepare specimens for visualizing the subcellular myosin subfragment and actin filaments, which employed localization of phytochrome A apoprotein (PHYA) by immuno- metal-contact liquid helium cryofixation immediately follow- cytochemistry in electron micrographs of pea hook cells. ing photolysis of caged ATP (Funatsu et al. 1993). In these Endogenous pea PHYA was specifically detected by experiments, apparatus for control of the ultraviolet light pulse immunogold-labeling in ultrathin sections, using PHYA- necessary for photolysis were constructed in the plunger of the deficient fun1-1 mutant (far-red unresponsive, Weller et al. cryofixation device (Funatsu et al. 1993). With further modifi- 1997) as a negative control. When etiolated seedlings were cations, our rapid-freezing device will be valuable for these exposed to continuous red light (cR) for 30 min, small aggrega- kinds of time-resolved investigations of various biological tions of immuno-gold appeared in the nucleus (Fig. 5A). Using responses. More generally, the automatic cryofixation device 5 nm immunogold particles, the aggregated form was seen in will be a powerful tool for future analyses of dynamic events, electron-dense areas of around 50 to 100 nm in diameter (Fig. for localization of proteins and water-soluble substances by 5B). This result is consistent with previous reports that PHYA immunocytochemistry, and for detecting specific elements by protein (Hisada et al. 2000) and GFP-PHYA fusion protein X-ray microanalysis. (Kircher et al. 1999) both show a speckled distribution within the nucleus under cR light. These observations also confirm the Materials and Methods intra-nuclear localization of PHYA, since in previous studies using optical microscopy it was difficult to prove that the Plant material speckling form of immuno-fluorescence associated with the Seeds of Arabidopsis thaliana (ecotype Landsberg erecta)were nucleus was truly appearing inside it. Further TEM studies of incubated on filter paper saturated with tap-water for 2 d at 23°C under the rapid translocation of PHYA will benefit greatly from the white light (fluorescent tube, FL40SW-B, Hitachi, Tokyo). Root tips high spatial and temporal resolution provided by the auto- were excised from germinating seeds by a razor blade and the root cap was mounted on the sample holder (Fig. 1A, a) which is brought into mated rapid-freezing device described here. direct contact with the cooled block for freezing (Fig. 1B, a and b). Seedlings of Arabidopsis were further cultured for 1 month under Metal-contact freezing technique vs. high pressure freezing continuous white light at 23°C. Stigmas were then excised from flow- method ers by a razor blade and the tip was mounted on the sample holder as Because of the high pressure, a lower cooling rate was above. acceptable and this method has therefore made it possible to Seeds of wild-type pea (Pisum sativum cv. Torsdag) and the iso- genic phyA-deficient fun1-1 mutant (for far-red unresponsive, Weller prepare considerably thicker and larger cryo-specimens (maxi- et al. 1997) were imbibed in water for 6 h in darkness and grown on mum thickness 600 mm by double-sided cooling) without for- vermiculite saturated with water at 23°C in darkness. Five-day-old mation of ice crystals (Sitte et al. 1987). A cooling rate can be seedlings were exposed to continuous red light for 30 min. Red light 892 Automated cryofixation device using liquid helium was obtained from fluorescent tubes (FL-20S Re-66; Toshiba, Tokyo) Electron microscopy filtered through 3-mm thick red acrylic plate (Acrylight K5-102; Mit- Ultrathin sections were prepared using an ultramicrotome (Sor- subishi Rayon, Tokyo), 3-mm thick scattering filter (Acrylight K5- vall® MT-6000, Du Pont Company, Wilmington, Delaware, U.S.A.). 001E; Mitsubishi, Rayon), and 3-mm thick white glass. The light The sections of Arabidopsis root tip and stigma were stained with ura- intensity measured at 660 nm was 55 mmol m–2 s–1. Immediately after nyl acetate and lead citrate, and examined in a TEM (H-7100, Hitachi the light irradiation, hook regions were excised from the epicotyl and Ltd., Tokyo). the epidermis was mounted on the sample holder as above. Immunolabeling Automated cryofixation The ultrathin sections of peas were subjected to immunolabeling Automated operation of the cryofixation device is illustrated in using polyclonal anti-pea phytochrome A apoprotein (PHYA) anti- Fig. 1. Prior to automated steps for cryofixation, the contacting sur- body (pAP) and gold-conjugated (particle size 5 or 10 nm) anti-rabbit face of the copper block (Fig. 1A, b) was polished, rinsed in acetone, IgG antibody (AuroProbe EM; Amersham Pharmacia Biotech., Upp- exposed to dry air and settled on the basement block (Fig. 1A, c) in the sala, Sweden). Immunolabeling was performed as follows: (1) sec- Dewar flask. When the copper block was handled, the block port (Fig. tions were treated in phosphate-buffered saline (PBS) containing 4% 1A, d) was positioned right above the copper block (Fig.1A, b). After bovine serum albumin (BSA) for 30 min to prevent non-specific bind- ° this operation, the plunger guide pipe (Fig. 1A, e) replaced with the ing; (2) incubation overnight at 4 C in anti-PHYA-antibody (final con- centration: 84 ng ml–1) in PBS; (3) a rinse in PBS containing 4% BSA, block port, sifting horizontally by 10 mm from the center of copper three rinses in PBS containing 0.05% Tween20 (Bio-Rad, Hercules, block (Fig. 1A, b). The outer Dewar flask (Fig. 1A, f) was filled with CA, U.S.A.) every 5 min, and a rinse in PBS containing 4% BSA for liquid nitrogen and the inner one (Fig. 1A, g) was filled with liquid 5 min; (4) incubation for 2 h in anti-rabbit IgG antibody; (5) six more helium up to the neck of the basement copper block (Fig. 1A, c) using rinses in PBS; and (6) treatment with 2% glutaraldehyde in distilled transfer tube (Fig. 1A, h) connected to the liquid helium tank. While water to fix the immunolabeling. Sections were stained with uranyl pouring the coolant, evaporating gas was allowed to escape through acetate. the valves (Fig. 1A, i). A fresh specimen mounted on the hollow of the specimen holder (Fig. 1A, a) was placed on the tip of the plunger (Fig. 1A, j) that was Acknowledgements inclined (Fig. 1A, arrow*) to enable this manual operation, and then returned to the vertical position (Fig. 1A, arrow**) before the start of We are grateful to James L. Weller for critical reading of this the rapid freezing steps. After turning on the switch button (Fig. 1A, k) manuscript, Hisafumi Ohtsuka (Hitachi, Ltd., Instrument Division) for of the controller (Fig. 1A, l), the following steps proceeded automati- developing the new technology, Shoukichi Matsunami and Wataru cally in sequence (Fig. 1B and 1C); namely, (1) the plunger (Fig. 1B, j) Moriya (Hitachi Advanced Research Laboratory, Technical Support was inserted into the guide pipe (Fig. 1B, e) and when the shutter (Fig. Center) for manufacturing of the device, and Sumiko Yabe for techni- 1B, m) of the guide pipe opened, warm air inside the guide pipe (Fig. cal supporting. We thank to James B. Reid for providing pea seeds, 1B, e) was ejected out from the device through the air escape (Fig. 1B, and Akira Nagatani and Hiroko Hanzawa for providing anti-phyto- n); (2) the sample on the holder (Fig. 1B, a) was rapidly frozen by con- chrome A antibodies. This work was supported by Hitachi Advanced tacting to the surface of the chilled copper block (Fig. 1B, b); (3) the Research Laboratory projects (B2023) and a grant from the Program plunger (Fig. 1C, j) rose to the initial position and the shutter (Fig. 1C, for Promotion of Basic Research Activity for Innovative Biosciences m) closed immediately; (4) the storage bottle (Fig. 1C, o) filled with to M. F. liquid nitrogen moved just under the plunger (Fig. 1C, j, arrow***) and the specimen on the holder (Fig. 1C, a) was immediately pushed References into liquid nitrogen; (5) the copper block (Fig. 1C, b and c) turned by one-eighth (arrows****) so that the clear surface was ready for the Butler, W.L., Norris, K.H., Siegelman, H.W. and Hendricks, S.B. (1959) Detec- next sample (Fig. 2). tion, assay, and preliminary purification of the pigment controlling photore- After eight samples were consecutively cryofixed, the upper sponsive development of plants. Proc. Natl. Acad. Sci. USA 45: 1703–1708. block (Fig. 1C, b) was taken out using the block holder through the Fujino, T. and Itoh, T. (1998) Changes in pectin structure during epidermal cell elongation in pea (Pisum sativum) and its implications for cell wall architec- block port (Fig. 1C, d). Then, a freshly polished copper block was ture. Plant Cell Physiol. 39: 1315–1323. replaced on the basement block, and the Dewar flasks were refilled Funatsu, T., Kono, E. and Tsukita, S. (1993) Time-resolved electron microscopic with coolant. analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP. J. Cell Biol. 121: 1053–1064. Freeze substitution and resin embedding Galway, M.E., Rennie, P.J. and Fowke, L.C. 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(Received June 8, 2001; Accepted July 12, 2001)