An Automated Device for Cryofixation of Specimens of Electron Microscopy Using Liquid Helium
Total Page:16
File Type:pdf, Size:1020Kb
Plant Cell Physiol. 42(9): 885–893 (2001) JSPP © 2001 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 pres- sure 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 nec- essary for preparation of multiple samples, and (4) significant reduction in running costs. First, we can put a great emphasis on extremely easy oper- ation of this automated cryofixation device. It is possible to carry out cryofixation of living samples without skill or prac- tice, 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.