Cryo-preservation for scanning electron microscopy avoids artefacts induced by conventional methods of specimen preparation

TISSUE & CELL 1986 18 (2) 305-311 © 1986 Longman Group Ltd

JOHN A. SARGENT

CRYO-PRESERVATION FOR SCANNING ELECTRON MICROSCOPY AVOIDS ARTEFACTS INDUCED BY CONVENTIONAL METHODS OF SPECIMEN PREPARATION Keywords: Scanning electron microscopy, cryo-preservation, fully hydrated tissues, surface waxes ABSTRACT. Cryo-preservation of tissues for scanning electron microscopy avoids artefacts associated with critical point drying and freeze-drying (solvent extraction and cell distortion). Motile specimens are arrested immediately, cells remain fully hydrated, delicate structures are undisturbed and conductive coating with metals or carbon is achieved at low temperatures.

The scanning electron microscope (SEM) has over the last 20 years permitted the examination of surface features of biological specimens at a resolution and depth of field far in excess of that obtainable with light optics. However, the environmental conditions to which specimens are exposed on the stage of an SEM are alien to living tissues and have demanded the adoption of special preparative methods to minimize physical distortion of hydrated material in the high vacuum and image aberrations due to specimen charging in the electron beam. These methods were designed to remove water from the specimen in a controlled manner before insertion into the microscope and to prevent charging by coating the specimen with a very thin conducting material, usually a metal. Critical point drying and freeze-drying have been the prefered techniques for the removal of water from tissues with the minimum of alterations in form and dimensions (Beckett et al., 1981; Boyde and Franc, 1981; Boyde and Maconnachie, 1979). Critical point drying involves dehydrating the specimen through a graded series of hygroscopic solvents such as ethanol,

Hexland Ltd, East Challow, Wantage, Oxfordshire OX12 9TF. 305

followed by the substitution of acetone or amyl acetate for the dehydrating agent. Subsequently the intermediate solvent is exchanged for liquid carbon dioxide under pressure and, after raising its temperature above its critical poin t the CO2 is bled off to the atmosphere. Thus during critical point drying the specimen is immersed in a number of solvents, including water if it is initially fixed in an aqueous solution. Any component which is soluble in one of these solvents is unavoidably removed from the specimen and delicate surface structures are liable to be swept away by inevitable agitations during handling in these liquids. The loss of epicuticular wax from the aerial surfaces of plants (Sargent, 1983) and the dislodgement of fungal spores from fruiting structures are examples of such serious artefacts associated with critical point drying. Freeze-drying might be expected to produce dehydrated specimens unaltered in form from their hydrated condition. Regrettably, however slowly water is removed by this method, soft tissues distort and reduce somewhat in volume. Such distortion of underlying cells can have a profound effect upon the orientation of surface structures subtended by these cells (Sargent, 1983). Conductive surface coatings are usually

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applied by evaporation from heated elec- incorporates a heater by which its temperatrodes, or more commonly for scanning ture can be raised and maintained at any electron microscopy, by sputtering in an preset level up to 323K (50°C). This enargon plasma. Although the latter method ables surface water films to be sublimed in a involves less generation of heat, specimen controlled manner or freeze-fracture sursurface temperatures do rise and can exceed faces to be etched. An evacuated prethe melting point of certain components chamber from which specimens can be such as wax. transfered directly to the microscope chamCryo-preservation avoids these problems. ber contains another cooled stage on which Water is not removed from specimens but is specimens can be freeze-fractured or coated maintained within them at a temperature at at low temperature. All the examples illuswhich its vapour pressure is virtually zero trated were sputter-coated with gold at (below 143K, -130°C). Specimens are co- approximately l l 0 K (-163°C). ated with conductive films at low temperaFig. 1 shows conidiophores of the mould ture and examined and photographed on a Aspergillus sp. which had been cultured on cooled stage. Neither chemical fixatives nor an agar plate. A small portion of the solvents are used. If only surface features fruiting mycelium was attached to a copper are to be observed cooling rates are unim- stub with conducting cement and quickly portant but rapid cooling minimizes internal plunged into nitrogen slush. This specimen, ice formation, an important consideration if like many fungi cultured on artificial media, freeze-etched faces are to be examined. supported a thin film of water. However, Dipping specimens into boiling cryogens after carefully observing sublimation of this such as liquid nitrogen is not recommended on the microscope stage at approximately because the evolved gas forms an insulating 190K (-83°C) foilowed by quickly cooling layer around them and the bubbling can to 88K (-185°C) and sputter-coating in the dislodge surface structures. Nitrogen slush pre-chamber, surface features could be at 63K (-210°C) obtained by evaporating observed in detail. While the vegetative liquid nitrogen under low pressure is a good hyphae have relatively smooth cell walls the general cryogen free from the fire hazard developing conidiophores are sculptured. associated with liquified hydrocarbons and The presence of a full component of conidia less costly than the freons. on the mature conidiophores demonstrates The micrographs shown in Figs 1-6 were the value of the technique not only in obtained using a Hexland CT 1000 cryo- maintaining the cells fully hydrated but also transfer system fitted to a Philips 505 SEM. 'in retaining detail and easily dislodged comThe specimen stage of the system is cooled ponents. by nitrogen gas at 88K (-185°C) and it Fig. 2 is a micrograph of a young cryo-

Fig. 1. Cryo-preserved developing conidiophores of Aspergillus sp. Cells are fully hydrated and conidia have not been dislodged, xlS00. Fig. 2. A newly hatched larva of Pieris brassicae frozen in the act of consuming its egg case. Threads of exuded silk have been preserved in situ. x200. Fig. 3. Hyphae of Erysiphe grarninis advancing over the surface of a leaf of wheat. Cells of both fungus and host are turgid and the epicuticular wax is perfectly preserved, x1500. Fig. 4. The head of Aleyrodes brassicae. Secreted wax covers virtually the entire surface except over the compound eye and the joints of the appendages, x300. Fig. 5. A larva of Aleyrodes brassicae. Wax is secreted as a peripheral fringe, x300. Fig. 6. The wax fringe of a larva ofAleyrodes brassicae. That which does not shed as sheets (Fig. 5) breaks away as crescent-shaped particles. ×4700.

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CRYO-PRESERVATIONFOR SEM preserved larva of the butterfly Pier& brassicae. It was one of a group of hatching larvae many of which were consuming their egg cases. The area of cabbage leaf on which the eggs had been laid was excised, stuck to a stub and cooled on the stage of the pre-chamber.' R a p i d freezing was not necessary and by cooling in this way the larvae were not disturbed. In addition the threads of silk which the larvae produce in abundance were retained in situ. Fig. 3 shows the surface of a cryopreserved wheat leaf over which the powdery mildew fungus Erysiphe graminis was advancing. The delicate hyphae are fully turgid, the positions of their transverse cell walls are evident and surface sculpturing has been retained. Epicuticular wax particles on the leaf surface have been preserved and, as far as can be judged, are not disorientated. Wax-free linear patches on the guard cells bounding the stomatal pore probably arise from the rhythmic changes in volume and surface area of these cells. Retention of wax on the surface of insects is demonstrated in Figs 4, 5 and 6. The head of the whitefly Aleyrodes brassicae is shown in Fig. 4. Wax is secreted through the cuticle in abundance by this insect and

covers virtually its entire surface except over the compound eyes and the joints of the appendages. The larva secretes wax as a fringe along its periphery. This evidently breaks away as sheets (Fig. 5) or as a crescent-shaped particles similar in size and shape to those present on the surface of the mature insect (Fig. 6). These examples demonstrate the potential of the cryo-preservation technique. Preparation ttmes are short, motility is arrested, natural levels of hydration are maintained, and internal morphology and interaction between organisms can be studied by freeze-fracture. Moreover, X-ray micro-analysis can be performed on intact or freeze-fractured specimens, an invaluable feature where the location of soluble and mobile elements is important. Future developments can be expected to include the progressive surface etching of cryopreserved specimens by the ion beam method. Acknowledgements I wish to thank Professor D. S. Smith for his encouragement and help in the preparation of this manuscript.

References Beckett, A., Porter, R. and Read, N. D. 1981.Low temperature scanningelectronmicroscopyof fungalmaterial.J. Microsc., 125, 193-199. Boyde, A. and Franc, F. 1981. Freeze-dryingshrinkageof glutaraldehydefixed liver.J. Microsc., 122,75-86. Boyde, A. and Maconnachie,E. 1979.Volumechangesduringpreparationof mouse embryonictissue for scanning electron microscopy.Scanning, 2, 149-163. Sargent, J. A. 1983. The preparation of leaf surfaces for scanningelectron microscopy: a comparative study. J. Microsc., 129, 103-110.