Loligo. - Journal of Cell ScienceSpecimens of Loligo vulgaris were immobilized by cutting off their arms and body and the statocysts were dissected out, fixed in 2% osmium tetroxide

J. Cell Set. 4, 223-239 (1969) 223

Printed in Great Britain

FREEZE-DRYING METHODS FOR THE

SCANNING ELECTRON-MICROSCOPICAL

STUDY OF THE PROTOZOON SPIROSTOMUM

AMBIGUUM AND THE STATOCYST OF THE

CEPHALOPOD MOLLUSC LOLIGO VULGARIS

A. BOYDE

Department of Anatomy, University College, Gotver Street, London, W.C. 1

AND V. C. BARBER

Department of Zoology, University Road, Bristol 8, England

SUMMARY

The present study concerns methods of preparing ciliated surfaces for direct examinationin the scanning electron microscope.

Air-drying methods provide good results with some ciliated structures but do not alwayspreserve the cilia of ciliated protozoons, although the pellicle is well preserved. Air-drying isnot suitable for certain epithelia because considerable shrinkage and tearing occur.

Freeze-drying methods, with or without pre-fixing, are described. These preserve the ciliain the protozoon Spirostomum in a fairly life-like position. There are some differences in theappearance of unfixed and fixed freeze-dried material—for example, the peristomial mem-branelles are not seen in the unfixed material. Freeze-drying again proved to be a bettermethod of preparing the sensory epithelium lining the statocyst of the cephalopod molluscLoligo, because it was successful in preventing the distortion due to shrinkage. The number ofhair cells, their orientation, and the area covered by the cells was determined for the macula.The crista was found to be asymmetrical.

INTRODUCTION

In our first scanning electron-microscopical account of the surface structure ofciliated epithelia a method of preparing these tissues was described that was bothsimple and rapid to use (Barber & Boyde, 1968). Although this method gave goodresults with many ciliated epithelia and other tissues, it was not found to be suitablefor all tissues. For example, the original methods were tried on several species ofprotozoons, but resulted in the almost complete removal of cilia, although thepellicle was often left quite beautifully displayed. Because of the shrinkage thatoccurs on air-drying, cracks and hence distortion occur in certain tissues. This was aparticular problem where the epithelium to be studied overlaid a substance such ascartilage that did not shrink to the same extent as the epithelium. An example of suchan epithelium is the receptor region in the statocyst of decapod cephalopods such asLoligo. Another situation where shrinkage was a problem was in epithelia wherethe cells were not firmly attached to each other by desmosomes or other attachmentstructures, and so became separated.

224 A- Boyde and V. C. Barber

In this present study, specimens of a protozoon(Spirostomumambiguum)v?ere chosenas test objects of particular delicacy in developing a more suitable and generallyapplicable method for the preservation of cilia in a life-like spatial position. Suchmethods would naturally be suitable for preparing any soft tissue surface, since theproblem involved in preserving cilia must be one of the most difficult that could beencountered in this field. Spirostomum was chosen as it is of a comparatively largesize and so is convenient to handle, and its structure is quite well known fromtransmission electron-microscopical studies (Daniel & Mattern, 1965; Finley, Brown& Daniel, 1964; Randall, 1957; Yagiu & Shigenaka, 1963). The statocyst of thecephalopod Loligo vulgaris was also studied as an epithelium where shrinkage is aparticular problem. The transmission electron-microscopical structure of cephalopodstatocysts is also quite well known (Barber, 1965, 1966a, b, 1968).

METHODS

The specimens of Spirostomum ambiguum used for freeze-drying were either usedlive or were fixed in 2% osmium tetroxide solution (phosphate buffered to pH 7-4,Millonig, 1961) or 6 vols. of this osmium solution to 1 vol. of a saturated solution ofmercuric chloride (following the method of Parducz, 1967). A considerable excess offixative was added to a small volume of water containing the live animals and thespecimens were fixed for approximately 2̂ —3 h.

For freeze-drying, drops of water containing the fixed or live animals were putinto small aluminium-foil 'boats' and these were immersed in a bath of 'Arcton 12'(dichlorodifluoromethane, I.C.I., Ltd.). The Arcton was cooled with liquid nitrogenand was kept just above its freezing point of — 155 °C. The frozen water drops weretransferred to larger aluminium ' boats' whilst still in the Arcton, and the contentsof the boats were frozen solid with liquid nitrogen. They were transferred in thiscondition to the surface of a cold probe inside the vacuum coating unit and freeze-dried, the complete cycle occupying some 18 h. The cold probe consisted of a smallmass of copper at the end of a large body of polytetrafluorethylene, there being asystem of tubes continuous between the parts. The copper was cooled by forcingliquid nitrogen through the tubes. The cooling was therefore very rapid, and theprobe could be cooled in a few seconds before placing the specimen on it, thus limitingthe time during which the condensation of atmospheric water vapour could occur onthe probe. The temperature of the probe was monitored with a thermocouple.Freeze-drying the specimens in the vacuum evaporator made it possible to give thespecimens a preliminary coating of carbon to stabilize them before exposing themto the atmosphere. This is an important point, because small freeze-dried organismsmay be hygroscopic.

Where some of the organisms remained adherent to the aluminium-foil 'boats',this portion of the boat was carefully cut out, and adapted onto the surface of astandard specimen holder. Other specimens were glued to the surface of the stubsusing a little of the glue that can be obtained by dissolving adhesive cellulose tape(Sellotape) in chloroform. The specimens were put back into the vacuum evaporator

Freeze-drying of cilia 225

and arranged to face at an angle of some 30° to a carbon source and 6o° to a goldsource, and were rotated continuously during the deposition of about 200 A of carbonand about 300 A of gold (see Barber & Boyde, 1968; Boyde, 1967). This prevents'charging' of the specimens caused by the electron probe.

For air-drying, the species initially used were Paramecium caudatum, Stentorpolymorphus and Spirostomum ambiguum, but Spirostontum was used for most of thestudy. Many fixatives were tried but the only one that gave any subsequent pre-servation of cilia was the mixture of Parducz (1967). After fixing for approximately3 h the specimens were dehydrated in graded 'Analar' acetone, solutions of 25, 50,75% and two changes of 100%, and were then pipetted on to specimen stubs andallowed to dry. Following drying the specimens were given the usual carbon and goldcoating in the vacuum evaporator.

Specimens of Loligo vulgaris were immobilized by cutting off their arms and bodyand the statocysts were dissected out, fixed in 2% osmium tetroxide solution (phos-phate buffered to pH 7-4, Millonig, 1961) and treated in the same way as air-driedprotozoa. After dehydration the specimens were bisected, air-dried, and stuck toviewing stubs with glue (Uhu glue, H. u M. Fischer, Buhl/Baden), the receptorsurface being uppermost. Some specimens of squid were anaesthetized in 2% urethanein sea water, and perfused with 4% neutral formaldehyde in sea water (Dr R. Martin,Stazione Zoologica, Naples, Italy, kindly prepared these specimens). The statocystswere dissected out and sent to London, England, immersed in fixative. They werefreeze-dried as before, the only difference being that isopentane was used as thequenchant.

All specimens were examined in a Cambridge Scientific Instruments Stereoscanscanning electron microscope operated at 10 kV, micrographs being recorded during(single) 100 s scans to produce virtually noise-free images. Stereoscopic-pair images(io° tilt) were recorded and later studied with the aid of a stereo-viewer (Hilger andWatts Stereometer) (see Boyde, 1967).

RESULTS

Spirostomum—freeze-dried material

Figure 1 shows the main surface features of the animal when prepared by freeze-drying. The peristomial groove (leading to the mouth) runs along much of the lengthof the animal. The cilia in this groove are arranged in groups to form peristomialmembranelles. The pellicle of the animal is composed of parallel ridges and grooves.Rows of cilia arise from the grooves (Daniel & Mattern, 1965; Finley et al. 1964;Yagiu & Shigenaka, 1963).

The appearance of freeze-dried specimens prepared by different methods wassimilar but there were differences of note. Cilia arise from the pellicular grooves inboth the fixed and unfixed specimens (Figs. 2, 6). Apart from the osmium tetroxide/mercuric chloride freeze-dried specimens (Fig. 4) the cilia of the fixed specimens aregenerally less smooth in appearance and they are often bent and are partly attachedto each other or to the pellicle (Figs. 2, 3). The cilia are much smoother and more

15 CeU Sci. 4

226 A. Boyde and V. C. Barber

'lifelike' in position in unfixed, freeze-dried specimens (Figs. 5, 6). It is not certainthat the cilia are exactly retained in their metachronal position but examination ofparts of the various figures shows that this is a possibility. The other feature to noteis the variable appearance of the pellicular surface. The pellicular ridges are quitepronounced in some specimens (Figs. 4, 6) whereas others are less obvious and somevery smooth surfaces were produced (Figs. 3, 5). Holes, which are probably freezingartefacts (they have been seen in other tissues such as muscle) are also seen, and aregenerally more obvious in unfixed specimens (Figs. 3, 5). Both the freeze- and air-dried OsO4/HgCl2 material (Figs. 4, 10) presented a nodular surface. It is likely thatthis result is caused by the large mitochondria and large granules that occur belowthe pellicular surface.

Transmission electron-microscopical studies have shown that the membranellesare arranged in 3 rows of about 12 cilia each, which are separated from the nextgroup by a peristomial fold, and there is further evidence of this in the arrangementof the microtubular arrays connected to the basal bodies of each cilium (Daniel &Mattern, 1965; Finley et al. 1964; Randall, 1957; Yagui & Shigenaka, 1963). Thesemembranelles are very well seen in fixed specimens (Fig. 7) but are not seen in theunfixed material, the cilia being separate (Fig. 8).

Spirostomum—air-dried material

Apart from the OsO4/HgCl2 fixed specimens the different ways of preparing air-dried specimens were unsuccessful, the pellicular ridges and grooves being well seenbut no cilia being present. However, specimens fixed by the Parducz method didhave cilia although they did not stand out in free space (Figs. 9, 10).

Loligo statocyst

The structure of the statocysts in cephalopods will not be given in detail here (seeBarber, 1968; Hamlyn-Harris, 1902; Ishikawa, 1924; Young, i960). Essentially, thestatocysts of decapod cephalopods such as Loligo consist of an epithelium, the staticsac, closely applied to the wall of a cartilaginous capsule. In octopods there is aperilymphatic space between the static sac and the cartilage, but this is absent indecapods. There are two main sensory regions; the macula, which is a flat plate ofcells overlaid by a crystalline statolith, and the crista, an elongated ridge of cells witha complicated orientation (see Barber, 1968).

The general appearance of air-dried material can be seen in Fig. 11, which alsoshows that a considerable amount of tearing occurs because of the shrinkage duringair-drying. This can be eliminated, or at least largely limited, by freeze-drying. Someidea of the amount of shrinkage which can occur can be gained from a comparison ofthe distance between the ciliary groups in Fig. 12 (air-dried) and Fig. 15 (freeze-dried).

Failure to remove the mucus layer covering wet epithelia inevitably means thatthe specimen surface consists of a varnish of dried mucus which obscures details ofinterest. Perfusion fixation fixes mucus in situ (Fig. 16). Better results can be obtainedby washing the epithelial surface before fixation, but we have been unable to achievereliable removal of mucus with any of a large variety of washing fluids that we have


tried. The statoliths were removed in the specimens used in these studies, but somecrystals remain (Figs. 11, 12, 13).

The receptor region in cephalopod statocysts is composed of hair cells and sup-porting cells. The hair cells bear numerous kinocilia (Figs. 12, 13)—up to 200 percell in Octopus (Barber, 1966 a)—and both they and the supporting cells bearnumerous microvilli (Fig. 13). It was not possible to count the number of hair groupsin the air-dried macula, but counts on the freeze-dried material gave a figure ofabout 1800 ciliated cells per macula. Again, although the general shape of the macula(Fig. 11) and the orientation of its ciliary groups could be determined from the air-dried material, a much clearer picture was given by the freeze-dried material, wherethe orientation of the long axis of each group was also determined (Figs. 14, 15).The crista of Loligo is asymmetrical (Fig. 16), thus resembling that of Eledone (Barber& Boyde, 1968) and Octopus (unpublished results).

DISCUSSION

Freeze-drying of Spirostomum is successful in preserving the cilia in free space.Parducz (1967) concluded that his special method of preparing protozoons was suchthat the cilia were preserved in their metachronal positions. Our results show thatthe Parducz method is much better than the other fixation procedures that weemployed on Spirostomum in fixing cilia in a position which is similar to the one theywould probably occupy when beating in metachronal waves. We would like toreserve comment on whether this conformation of cilia is 'real', or is a special resultof the Parducz method, but we should like to observe that the Parducz fixationprocedure leaves the cilia in a position which is similar to the position of the cilia inlive, freeze-dried specimens. Incidentally, the Parducz method also leaves the ciliain a condition where they can be preserved after air-drying.

The fusion of rows of cilia to form the peristomial membranelles is absent inunfixed specimens. We are inclined to believe that some feature of the quenching andfreeze-drying process has caused the separation of the members of these rows (i.e.that they are functionally attached), but it must be noted that the various conventionalelectron-microscopical studies which have demonstrated the existence of the mem-branelles (Daniel & Mattern, 1965; Finley et al. 1964; Yagui & Shigenaka, 1963) alsoemployed fixed material. The possibility that the fusion of cilia to form membranellesin fixed material is a fixation artefact, rather than that their separation is a freezingartefact, cannot be excluded. Grim (1966, 1967), in his transmission electron-microscopical study of Euplotes using a critical-point freeze-drying procedure onfixed, partly disrupted specimens, also obtained membranelles.

Freeze-drying techniques again proved their value in the study of the statocyst ofLoligo. The results show that it should be possible to count the exact number ofreceptor cells in any ciliated epithelium and a preliminary result of such a count onthe rabbit olfactory mucosa has already been reported (Barber & Boyde, 1968). Suchcounts could be correlated with counts of the number of nerve fibres going to, orleaving, the receptors, for a functional analysis. Another useful possibility is that

15-2


complete receptor areas could be mapped out in other animals as has been done inLoligo. For example, in Octopus the basal feet borne on the basal bodies of the ciliapoint towards the periphery of the macula, and it has been suggested that movementof the cilia in the direction of their basal feet is excitatory (Barber, 1968). This isanalogous to the situation in the acoustico-vestibular systems of vertebrates (Lowen-stein & Wersall, 1959; Lowenstein, Osborne & Wersall, 1964; Flock & Duvall, 1965).Any conclusion about the squid statocyst must be uncertain without transmissionelectron-microscopical information, but if the basal feet do all point towards theperiphery, and movement of cilia in that direction is excitatory, a complete map ofthe electrophysiological responses of the cells is now available.

We have found no evidence for or against the existence of a cupula on the cristaof the cephalopod species that we have examined. The structure obviously occupiesthe same spatial domain as the mucus-containing fluid of the statocyst, and has onlybeen reported from light-microscopic studies (as for example by Young, i960, inOctopus and Young, 1968, in Sepia), where its fragility has been noted. It is probablethat a cupula, if present, would be removed along with the bulk of the mucus in thepreparative procedures that we have used, since these were aimed at washing awaythe mucus. We have already noted that perfusion fixation tends to fix the mucus insitu, and our unpublished scanning electron-microscopical observations have shownthat the cupula is preserved by perfusion of fixative into the semi-circular canals ofan elasmobranch fish.

The asymmetry of the crista in Loligo resembles that of the other cephalopods sofar studied, but no physiological evidence of its function has been reported. However,electrophysiological recordings from the middle crista nerve supplying the longi-tudinal portion of the crista in Octopus do show that elements are present that areunidirectional in response (Maturana & Sperling, 1963).

We would like to thank Professor J. Z. Young for his active assistance and advice. Thevisits to the Stazione Zoologies, Naples, Italy, for the collection of cephalopods, were partlyfinanced by a grant to Professor Young from the U.S. Office of Aerospace Research. Wewould like to thank Dr R. Martin for the perfused cephalopod specimens. We should also liketo thank Mr H. Coates, Mr R. Sampson, Mr M. Warrell and Dr K. S. Lester for their help inconstructing the vacuum evaporation unit and cold probe assembly used in these studies, andDr M. A. Sleigh and Miss M. Nicholson, who provided us with all our specimens of liveprotozoa. The scanning electron microscope used in this study (Department of Anatomy,University College London) was provided by a grant from the Science Research Council(U.K.).

REFERENCESBARBER, V. C. (1965). Preliminary observations on the fine structure of the Octopus statocyst.

J. Microscopie 4, 547-550.BARBER, V. C. (1966a). The fine structure of the statocyst of Octopus vulgaris. Z. Zellforsch.

mikrosk. Anat. 70, 91-107.BARBER, V. C. (19666). The morphological polarisation of kinocilia in the Octopus statocyst.

J. Anat. 100, 685-686.BARBER, V. C. (1968). The structure of mollusc statocysts, with particular reference to

cephalopods. Symp. zool. Soc, hand. (In the Press.)


BARBER, V. C. & BOYDE, A. (1968). Scanning electron microscopical studies of cilia. Z.Zellforsch. mikrosk. Anat. 84, 269—284.

BOYDE, A. (1967). A single-stage carbon-replica method and some related techniques for theanalysis of the electron microscope image. Jl R. microsc. Soc. 86, 359—370.

DANIEL, W. A. & MATTERN, C. F. T. (1965). Some observations on the structure of theperistomial membranelle of Spirostomum ambiguum. J. Protozool. 13, 14-27.

FINLEY, H. E., BROWN, C. A. & DANIEL, W. A. (1964). Electron microscopy of the ectoplasmand infraciliature of Spirostomum ambiguum. J. Protozool. 11, 264-280.

FLOCK, A. & DUVALL, A. J. (1965). The ultrastructure of the kinocilium of the sensory cellsin the inner ear and lateral line organs. J. Cell Biol. 25, 1-8.

GRIM, J. N. (1966). Isolated ciliary structures of Euplotes patella. Expl Cell. Res. 41, 206-210.GRIM, J. N. (1967). Ultrastructure of pellicular and ciliary structures of Euplotes eurystomus.

J. Protozool. 14, 625-633.HAMLYN-HARRIS, R. (1903). Die Statocysten der Cephalopoden. Zool.Jb. Anat. 18, 327-358.ISHIKAWA, M. (1924). On the phylogenetic position of the cephalopod genera of Japan based

on the structure of statocysts. J. Coll. Agric. Imp. Univ., Tokyo 7, 165-210.LOWENSTEIN, O., OSBORNE, M. P. & WERSXLL, J. (1964). Structure and innervation of the

sensory epithelium of the labyrinth in the thornback ray (Raja clavata). Proc. R. Soc. B160, I-I2.

LOWENSTEIN, O. & WERSALL, J. (1959). Functional interpretation of the electron microscopicstructure of the sensory hairs in the cristae of the elasmobranch Raja clavata in terms ofdirectional sensitivity. Nature, Lond. 184, 1807-1808.

MATURANA, H. R. & SPERLING, S. (1963). Unidirectional response to angular accelerationrecorded from the middle cristal nerve in the statocyst of Octopus. Nature, Lond. 197,815-816.

MILLONIG, G. (1961). Advantages of a phosphate buffer for OsO4 solutions in fixation. J. appl.Phys. 32, 1637.

PARDUCZ, B. (1967). Ciliary movement and coordination in ciliates. Int. Rev. Cytol. 21, 91-128.RANDALL, J. T. (1957). The fine structure of the protozoan Spirostomum ambiguum. Symp.

Soc. exp. Biol. 10, 185-198.YAGIU, R. & SHIGENAKA, Y. (1963). Electron microscopy of the longitudinal fibrillar bundle

and the contractile fibrillar system in Spirostomum ambiguum. J. Protozool. 10, 364-369.YOUNG, J. Z. (i960). The statocysts of Octopus vulgaris. Proc. R. Soc. B 152, 3-29.YOUNG, J. Z. (1968). The brain of Octopus vulgaris. (In the Press.)

(Received 22 April 1968)


Fig. 1. OsCXj-fixed, freeze-dried Spirostomum. Note the pellicular ridges andperistomial groove (arrow).Fig. 2. Higher magnification of a specimen prepared as in Fig. 1. The cilia stand awayfrom the cell in free space.Fig. 3. Higher magnification of the part outlined in Fig. 2. Note the somewhat bentnature of the cilia and the holes in the pellicular surface (arrows), which are probablya freezing artefact.

Freeze-drying of cilia


Fig. 4. Spirostomum fixed in osmium tetroxide/mercuric chloride and freeze-dried.Note the nodular surface of the pellicle and the obvious pellicular ridges. The ciliaare smoothly curved.Fig. 5. Unfixed freeze-dried Spirostomum, partly denuded of cilia in preparation. Thespecimen is almost completely extended. Note the smooth surface of the pellicle.Fig. 6. Unfixed freeze-dried Spirostomum. Note the smooth curves of the cilia and themore irregular surface of the pellicle.


234 A- Boyde and V. C. Barber

Fig. 7. Fixed freeze-dried Spirostomum showing the peristomial membranelles(arrow).Fig. 8. Unfixed freeze-dried Spirostomum. Note that the peristomial membranellesare not preserved with this method of preparation.Fig. 9. An air-dried osmium tetroxide/mercuric chloride fixed specimen of Spiro-stomum. The pellicular ridges are well seen although the animal has contractedconsiderably.Fig. 10. Higher magnification of part of Fig. 9 to show that cilia are preserved,although they adhere to the pellicular surface. This is the only air-drying methodthat we have tried that preserves cilia.



Fig. 11. The macula of the statocvst of Loligo from a fixed, air-dried specimen. Notethe tearing of the epithelium.Fig. 12. Higher magnification of the outlined part of Fig. 11 to show the ciliary groupsin more detail. Note the adherent statolith crystals (arrows).Fig. 13. Higher magnification of part of Fig. 11. Note the individual cilia in thegroups (k) and microvilli (mv).



Fig. 14. Fixed, freeze-dried specimen of the macula {mac) and crista (cr) in thestatocyst of Loligo. Mucus obscures part of the detail of the macular surface.Fig. 15. A higher magnification of part of a macula of Loligo, prepared as Fig. 14.The number of ciliary groups can easily be counted in such preparations.Fig. 16. Portion of the crista of a fixed, freeze-dried statocyst of Loligo. Mucusobscures some of the details. Each of the ciliary groups is composed of up to 200kinocilia (arrows).

Freeze-dfying of cilia 239

Documents

Loligo. - Journal of Cell ScienceSpecimens of Loligo vulgaris were immobilized by cutting off their arms and body and the statocysts were dissected out, fixed in 2% osmium tetroxide