An apparatus for preparing frozen-etched specimens for electron microscopy

An apparatus for preparing frozen.etched specimens for electron microscopy" received19July 1971 A W Robards and M H Cooper, Departmentof Biology, Universityof Yorkand NGN Ltd, Church,Accrington, England

Freeze-etching is a method which allows the internal structure of biological specimens to be studied by electron microscopy after minimal chemical pre-treatment ; thus the problem of artifacts is, to some extent, reduced. The technique requires that the specimens are precisely maintained at low temperature and high vacuum while the fracturing, etching, and replicating processes are carried out. The freeze-etching unit described here allows a wide range of different biological structures to be studied. The design criteria are considered and recent results illustrated.

Introduction

Freeze-etching was introduced as a routine preparation technique for electron microscopy by Moor et alI. It is a method which enables replicas to be obtained from the surfaces of frozen biological specimens. In essence, there are six distinct stages during freeze-etching: (1) Pre-treatment with chemicals to aid preservation of the tissue or to minimize freezing damage; (2) Freezing; (3) Fracturing; (4) Etching; (5) Evaporation of replica onto exposed surface; (6) Cleaning of replica. Stages 3-5 require special apparatus for their accomplishment; it is the purpose of this article to describe such a machine. The principle upon which freeze-etching is based is that most biological specimens, if treated suitably, can be frozen with very little resultant damage to structure even at the finest level of organization. A frozen specimen can be fractured and, by so doing, the internal structure of cells and other biological units will be revealed. By carefully controlling both vacuum and specimen temperature it is possible to sublime ice away from nonaqueous parts of the specimen, thus leaving such parts standing slightly above the general level of the surface. This sculptured face can then be replicated by shadowing it with an evaporated heavy metal which is reinforced with a layer of carbon evaporated from directly above. After removal from the apparatus any organic debris is dissolved away from the replica which is then ready to be examined in a transmission electron microscope.

F r o m the biological standpoint freeze-etching offers a num bet of advantages over more conventional methods of specimen preparation: large areas of membrane surfaces are exposed (a feature poorly presented in sectioned material); the preparation time can be impressively short; and, possibly of greatest significance, the amount of chemical treatment is at worst greatly reduced, and at best eliminated, as compared with other preparation methods. This means that the problems of erroneous interpretation due to artifacts are reduced. It does not, however, mean that the artifact problem is eliminated, and it is becoming increasingly clear that freeze-etching gives rise to its own unique artifacts which will need investigation and clarification to enable results to be properly interpreted and understood.

Pre-treatments are sometimes used to facilitate freezing. The most common form of treatment is with a solution of glycerol which enables freezing to be effected without ice-crystal damage. The basic requirement for freezing is that it should not give rise to large, damaging ice-crystals within the specimen. In the majority of cases this has meant very rapid freezing by immersing the specimen in a solvent bath such as dichlorodifluoromethane (Arcton, Freon) which is cooled with liquid nitrogen; this will give a heat loss of better than 100°C per sec. Once the specimen has been frozen it is ready for fracturing, at which stage it is usually transferred to the cold-stage of the apparatus. *Paper presented at the "Conference on Vacuum Equipment" arranged by the Vacuum Group of the Institute of Physics at the University of Sussex, 5-6 April, 1971. tNow at Bolton College of Education (Technical), Bolton, England.

Figure 1. An NGN FE600 freeze-etching unit.

Vacuum/volume 211number 10. PergamonPress LtdlPrintedin GreatBritain

465

A W Robards and M H Cooper: An apparatus for preparing frozen-etched specimens for electron microscopy Readers who would like to know more of the technique in general are referred to the papers of Koehler =, Moor 3 and Robards and Parish 4. The remainder of this article will deal with the freeze-etching machine itself. There are four major systems for consideration: (1) Vacuum; (2)Cryogenics; (3) Microtome; (4)Evaporation units. These will be discussed one-by-one. The general appearance of the N G N FE600 is illustrated in Figure 1. This machine has been derived from a freeze-etching machine which was designed and built in the Astbury Department of Biophysics, University of Leeds, under the supervision of Professor R D Preston, FRS. V a c u u m system The vacuum system of the FE600 is conventional and incorporates usual principles of high vacuum design. In particular, the number of spindles passing from air to high vacuum has been reduced to a minimum. The general arrangement of the vacuum components is shown in Figure 2. The work-chamber comprises a stainless steel closed cylinder, 18 ~ in. inner dia× 16 in. high which is sealed to the stainless steel base plate by an O-ring. The chamber is pumped by a 6 in. bore diffusion pump having an unbaffied speed of 650 l./sec and ol:erating through a 6 in. dia cold trap and butterfly valve. The diffusion pump is backed by a single stage rotary pump with a displacement of 371 l./min. Appropriate magnetic isolation and air admittance valves are provided to render the system "failsafe". A phosphorus pentoxide trap is fitted in the line to the rotary pump to remove water vapour. The liquid nitrogen

cooled cold trap produces a pumping speed for water of 1750 l./sec at the base plate compared with a speed of 300 l./sec for air. The backing tank allows the rotary pump to be switched off during replica evaporation so that there will be absolutely no mechanical vibration at this stage. Vacuum condition is monitored by Pirani heads positioned adjacent to the phosphorus pentoxide trap and in the backing tank, and by a Penning head in the work-chaml:er. Vacuum requirements for freeze-etching are, unfortunately, imprecisely known. Two major considerations arise: speed of operation and specimen contamination. The "productivity" of the technique relies upon the speed with which specimens may be processed. Thus, for any given vacuum, the pumping speed is limiting. As both specimen head and microtome assembly are cooled during operation, they act as condensing surfaces when the work-chamber is brought to atmospheric pressure; this means that water vapour will need to be pumped during the next cycle, so reducing pumping speed. The problem may be minimized eitEer by drying all surfaces prior to evacuation or by preventing condensation. In the FE600 the latter course is adopted by purging all lines of liquid nitrogen before air admittance to the work-chamber. In these circumstances it is possible to achieve a stable vacuum of 10-e torr in 30 min. Regarding specimen contamination, it is clear that both vacuum and specimen temperature will be contributory parameters. In practice, the cold microtome knife head is used as an additional local cold trap which, at --190°C will trap molecules more effectivelY than the specimen at -- 100°C. Using these temperatures, and a vacuum of l0 -e torr, contamination of the specimen

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A W Robards and M H Cooper: An apparatus for preparing frozen-etched specimens for electron microscopy during the interval between fracturing and replicating does not appear to be a problem. However, experiments in the laboratory of the senior Author indicate that a worse vacuum o r lower specimen temperature can result in contamination artifacts in the final replica. It is, therefore, imperative that the workchaml:er is always maintained clean and that fracturing, etching, and replication are only carried out once high vacuum has been obtained. At the present time 10 -8 torr appears adequate for most purposes but it seems highly probable that further investigations will indicate the necessity for higher vacuum. This may be obtained using a good quality diffusion pump system or, alternatively, by employing a turbomolecular pump. In the latter case it has not yet been shown whether the slight vibration associated with such a pump will be acceptable during the deposition of high resolution replicas. Cryogenic

economy of liquid nitrogen consistent with maintaining a knife temperature of about --190°C. Specimen temperature control is far more critical than that of the knife. Valve D is opened and shut by a temperature controller which is actuated by a platinum resistance element mounted in the base of the specimen table (Figure 4); actual temperature obtained is monitored by a further element positioned immediately below the specimen. The specimen itself is mounted on a small disc of copper foil which has been punched to the shape of a flat-topped cone; this provides an excellent compromise between the various requirements for the specimen support (Robards and Parish% The specimen temperature control system is accurate to ±0.1 °C; a small heating element around the internal specimen table body (Figure 4) is also incorporated in the control circuit to facilitate smooth and rapid temperature adjustments and, when required, to allow rapid warm-up of the specimen. To ensure that the liquid nitrogen pipe-work within the chamber stays dry and free from condensation at the end of each cycle, dry air can be blown through the supply pipes. By operating switches to open valves A and/or B a compressor is switched on and draws air through a column of molecular sieve drying agent before forcing the air through the supply pipes, so purging them of nitrogen. As mentioned above, C and D cannot be opened while A and B are open. A most important aspect of the cryogenic side of freezeetching is rapid and precise specimen temperature control.

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Liquid nitrogen is supplied to the machine from a pressurized Dewar vessel; safety valves preclude excessive pressurization. The liquid nitrogen supply is controlled through a circuit containing electro-magnetic valves (Figure 3). The valves are interlocked so that nitrogen cannot be fed to knife or specimen while the purging lines are open, or v i c e v e r s a . Knife cooling is effected by opening valve C which allows liquid nitrogen to flow under the control of E which is opened and shut by an electronic timing device set to give maximum

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Figure 3. The cryogenic system. 467

A W Robards and M H Cooper: A n a p p a r a t u s for preparing f r o z e n - e t c h e d specimens for electron m i c r o s c o p y SPECIMEN COPPER BLOCK

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Figure 4. The specimen table. F u r t h e r , s p e e d during the cooling a n d w a r m i n g phases, t o g e t h e r w i t h the time for required v a c u u m a t t a i n m e n t , d e t e r m i n e s the total cycle time. S o m e characteristic cycle p a r a m e t e r s are s h o w n in Table 1. T a b l e 1. Characteristic parameters obtained during a freeze-etching cycle using the FE600 Time Cooling/warming "Work" (min) State of vacuum Specimen Knife

0

Atmospheric pressure

-- 135°C

Ambient Load specimen, start evacuation cycle, adjust specimen controller to -- 100°C

2 4 6 8 10 12 14 16 18

10 3torr

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10-Storr

100°C

- 190°C

Preliminary "rough" cuts with microtome

20 22 24 26

10-~ torr

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-- 1 9 0 ° C

Final cuts with knife, position knife specimen, allow time for etching

28

468

30

10-s torr

-- 100°C

-- 190°C Finish etching by evaporating replica, start warming knife

32 34

10 5 torr

-- 100°C

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36

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-- 135°C

Leak air through drying column into dome Ambient Remove specimen, reload with prealigned evaporation jigs, load new specimen

38 40

Atmospheric

- 135°C

Ambient

Microtome

Start evacuating for new cycle

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The r e q u i r e m e n t s for p r o d u c i n g a fracture t h r o u g h the specim e n during the freeze-etching process are one o f the m o r e controversial areas o f the technique. Various workers have used different m e t h o d s ranging f r o m shearing the specimen before placing it u n d e r vacuum, t h r o u g h blades o n the ends o f m o v a b l e r o d s passing t h r o u g h v a c u u m seals, t o relatively c o m p l i c a t e d m i c r o t o m e s (see R o b a r d s et aP, a n d R o b a r d s a n d Parish'). While simple m e t h o d s will u n d o u b t e d l y suffice for m a n y types o f specimen, "difficult" objects m a y require m o r e careful a t t e n t i o n before a fracture plane can be p r o d u c e d w h i c h will be satisfactory for replication. The great majority o f specimens can be dealt with using a device which has m o r e precision a n d control t h a n a blade o n the e n d o f a stick, a n d yet is less sophisti-

A W Robards and M H Cooper: An apparatus for preparing frozen-etched specimens for electron microscopy The knife body is hollow so that it may be cooled by pumping liquid nitrogen through it. This body is suspended from two arms which, with the body and upper suspension block, form a parallelogram; thus the knife remains parallel to the base plate during the fracturing process. The actual cutting edge is a stainless steel razor blade which is firmly clamped to the knife body. A platinum resistance element located in the body immediately behind the blade allows temperature measurement which is indicated on the same meter as specimen temperature. Vertical movement of the knife is provided by a control shaft which is held rigidly by the control shaft block (Figure 5). The block also carries a guide pillar which prevents the upper suspension block from rotating. The control shaft passes through the base plate where it is sealed with an O-ring. The lower end of the shaft carries a 40 thread/in, micrometer thread which passes through the control nut. This nut has two gear wheels attached to it: a bevel gear; and a 100 tooth worm wheel. A lever on the front of the machine engages a hand wheel drive to either bevel or worm gear, giving 0.32 mm and 3.2/~m advance per complete rotation respectively. Even finer control of the depth of cut is obtained by heating a brass block which is

cated and costly than a fully engineered ultramicrotome. In particular it is worth noting that there is usually no necessity for the blade to retract from the specimen surface during the return stroke of the knife; this is because the fracture plane lies below the horizontal plane of movement of the blade edge. The knife assembly on the FE600 is basically a cooled razor blade suspended by strip hinges from an adjustable-height pillar and driven backwards and forwards across the specimen by solenoids (Figure 5). The solenoids are powered by a transistorized supply; by controlling the bias on the transistors the coil current, and therefore magnetic field, can be varied and so move the plunger to which the knife is connected. A small current remains applied to each coil even if the other is receiving full power; this provides smooth and progressive control. A push button on the control panel can completely over-ride the normal controlling potentiometer, and thus allows a very rapid forward movement of the knife to be obtained. The advantage of the solenoid system is that there are no moving spindles passing between high vacuum and air. The potential source of difficulties from outgassing solenoid coils has not occurred in practice.

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469

A W Robards and M H Cooper: An apparatus for preparing frozen-etched specimens for electron microscopy be overcome by using an electron beam gun to evaporate more refractory materials with finer grain crystallization diameters. In this way replica resolution of about 1.0 n m is possible, although precautions must be taken to avoid excessive electron, ion, or heat radiation damage to the specimen. The FE600, as described above, is a machine which is simple, effective, and productive. Its complexity is no greater than allows a full range of biological specimens to be processed satisfactorily; its simplicity has not been obtained at the expense of vacuum, cryogenic, and mechanical requirements that are necessary for reliable and routine use. As freeze-etching becomes increasingly accepted as a preparation method for electron microscopy, machines similar to the one described here will be essential for ensuring that all parameters during processing are under control. Further, attachments such as multiple specimen holders and double replica devices will extend both the productivity and scientific value of the technique. Already freeze-etching has provided new and valuable

located between the base plate and the control nut. Expansion of the block forces the control shaft down, so lowering the knife. A platinum resistance element located in the block measures its temperature which is displayed on a meter calibrated directly in terms of depth of cut; each division on the meter corresponding to a knife advance of 30 nm.

Evaporation units When fracturing and etching have been completed, it remains to shadow the specimen and to deposit a strengthening replica film. Virtually all freeze-etching so far carried out has been effected using platinum as the shadowing metal and carbon as the supporting film. The advantages of using these two elements are that they may be easily and consistently evaporated; resolution as limited by the replica is tolerably good (of the order of 3.0 rim); and the elements are not affected by the various solutions employed during the cleaning stages.

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The two evaporation sources are identical, being simple assemblies to allow a carbon arc to be struck (Figure 6). They are mounted on aluminium pillars which also act as the earth returns. The live carbon holder slides in a ball bushing and is lightly sprung to keep the rods in contact. An adjustable shield is provided to direct the evaporant onto the specimen and to reduce chamber contamination. Platinum is evaporated by winding a small coil of thin platinum wire around the points of the adjacent carbon rods. This simple system gives quite adequate and reproducible results without involving problems of radiant heat damage to the specimen. Unfortunately, the limitation to resolution precludes the full potential of the freeze-etching method from being achieved. The problem may

470

information and has acted as invaluable supporting evidence in conjunction with "conventional" sectioning methods. Figures 7 and 8 are micrographs from replicas of frozen etched surfaces which illustrate some of the features which makes this technique important in its own right.

References 1 H Moor, K Muhlethaler, H Waldner and A Frey-Wyssling,J biophys biochem Cytol, 10, 1961, 1. J K Koehler, Adv biol medPhys, 12, 1968, 1. a H Moor, lnt Rev Cytol, 25, 1969, 391. 4 A W Robards and G R Parish, Lab Pract, 1971 (In press). 5 A W Robards, W R Austin and G R Parish, Proc 7th Congr E M (Grenoble), 1, 1970, 447.

A W Robards and M H Cooper: An apparatus for preparing frozen-etched specimens for electron microscopy

Figures 7 and 8. Micrographs of frozen-etched specimens. In each case the direction of metal shadowing is from the top of the page. The prints are directly from the original negatives; thus, the "shadows" appear light. Figure 7. Part of the stigma of Petunia spp. The significant features are the large areas of membrane surfaces which have been exposed, and the clear demonstration of a vesicle or small vacuole (VE) fusing with a vacuole (V). The different nature of membrane surfaces can be seen (ER--endoplasmic reticulum; P l - - p l a s -

malemma). The cell wall (CW) separates two adjacent cells, x 21,500. Figure 8. A h u m a n erythrocyte (red blood cell). The fracture has passed over, and then through, the membrane. Different views of membranes have been obtained : a smooth surface (MS) typical of the outer membrane surface; and a particulate face thought to be produced by the fracture running through the membrane. The contents of the erythrocyte (EC) are revealed where the membrane has been completely removed, while the cell is surrounded by the frozen solution (S) in which the erythrocytes were suspended, x 18,000.

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