J. Cell Set. 4, 223-239 (1969)
Printed in Great Britain
223
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 examination
in the scanning electron microscope.
Air-drying methods provide good results with some ciliated structures but do not always
preserve the cilia of ciliated protozoons, although the pellicle is well preserved. Air-drying is
not suitable for certain epithelia because considerable shrinkage and tearing occur.
Freeze-drying methods, with or without pre-fixing, are described. These preserve the cilia
in the protozoon Spirostomum in a fairly life-like position. There are some differences in the
appearance of unfixed and fixed freeze-dried material—for example, the peristomial membranelles are not seen in the unfixed material. Freeze-drying again proved to be a better
method of preparing the sensory epithelium lining the statocyst of the cephalopod mollusc
Loligo, because it was successful in preventing the distortion due to shrinkage. The number of
hair 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 of
ciliated epithelia a method of preparing these tissues was described that was both
simple and rapid to use (Barber & Boyde, 1968). Although this method gave good
results with many ciliated epithelia and other tissues, it was not found to be suitable
for all tissues. For example, the original methods were tried on several species of
protozoons, but resulted in the almost complete removal of cilia, although the
pellicle was often left quite beautifully displayed. Because of the shrinkage that
occurs on air-drying, cracks and hence distortion occur in certain tissues. This was a
particular problem where the epithelium to be studied overlaid a substance such as
cartilage that did not shrink to the same extent as the epithelium. An example of such
an epithelium is the receptor region in the statocyst of decapod cephalopods such as
Loligo. Another situation where shrinkage was a problem was in epithelia where
the cells were not firmly attached to each other by desmosomes or other attachment
structures, and so became separated.
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A- Boyde and V. C. Barber
In this present study, specimens of a protozoon(Spirostomumambiguum)v?ere chosen
as test objects of particular delicacy in developing a more suitable and generally
applicable method for the preservation of cilia in a life-like spatial position. Such
methods would naturally be suitable for preparing any soft tissue surface, since the
problem involved in preserving cilia must be one of the most difficult that could be
encountered in this field. Spirostomum was chosen as it is of a comparatively large
size and so is convenient to handle, and its structure is quite well known from
transmission electron-microscopical studies (Daniel & Mattern, 1965; Finley, Brown
& Daniel, 1964; Randall, 1957; Yagiu & Shigenaka, 1963). The statocyst of the
cephalopod Loligo vulgaris was also studied as an epithelium where shrinkage is a
particular problem. The transmission electron-microscopical structure of cephalopod
statocysts is also quite well known (Barber, 1965, 1966a, b, 1968).
METHODS
The specimens of Spirostomum ambiguum used for freeze-drying were either used
live 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 of
mercuric chloride (following the method of Parducz, 1967). A considerable excess of
fixative was added to a small volume of water containing the live animals and the
specimens were fixed for approximately 2^—3 h.
For freeze-drying, drops of water containing the fixed or live animals were put
into 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 nitrogen
and was kept just above its freezing point of — 155 °C. The frozen water drops were
transferred to larger aluminium ' boats' whilst still in the Arcton, and the contents
of the boats were frozen solid with liquid nitrogen. They were transferred in this
condition to the surface of a cold probe inside the vacuum coating unit and freezedried, the complete cycle occupying some 18 h. The cold probe consisted of a small
mass of copper at the end of a large body of polytetrafluorethylene, there being a
system of tubes continuous between the parts. The copper was cooled by forcing
liquid nitrogen through the tubes. The cooling was therefore very rapid, and the
probe could be cooled in a few seconds before placing the specimen on it, thus limiting
the time during which the condensation of atmospheric water vapour could occur on
the probe. The temperature of the probe was monitored with a thermocouple.
Freeze-drying the specimens in the vacuum evaporator made it possible to give the
specimens a preliminary coating of carbon to stabilize them before exposing them
to the atmosphere. This is an important point, because small freeze-dried organisms
may 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 a
standard specimen holder. Other specimens were glued to the surface of the stubs
using 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 gold
source, and were rotated continuously during the deposition of about 200 A of carbon
and 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, Stentor
polymorphus and Spirostomum ambiguum, but Spirostontum was used for most of the
study. Many fixatives were tried but the only one that gave any subsequent preservation of cilia was the mixture of Parducz (1967). After fixing for approximately
3 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 and
allowed to dry. Following drying the specimens were given the usual carbon and gold
coating in the vacuum evaporator.
Specimens of Loligo vulgaris were immobilized by cutting off their arms and body
and the statocysts were dissected out, fixed in 2% osmium tetroxide solution (phosphate buffered to pH 7-4, Millonig, 1961) and treated in the same way as air-dried
protozoa. After dehydration the specimens were bisected, air-dried, and stuck to
viewing stubs with glue (Uhu glue, H. u M. Fischer, Buhl/Baden), the receptor
surface being uppermost. Some specimens of squid were anaesthetized in 2% urethane
in sea water, and perfused with 4% neutral formaldehyde in sea water (Dr R. Martin,
Stazione Zoologica, Naples, Italy, kindly prepared these specimens). The statocysts
were dissected out and sent to London, England, immersed in fixative. They were
freeze-dried as before, the only difference being that isopentane was used as the
quenchant.
All specimens were examined in a Cambridge Scientific Instruments Stereoscan
scanning 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 and
Watts Stereometer) (see Boyde, 1967).
RESULTS
Spirostomum—freeze-dried material
Figure 1 shows the main surface features of the animal when prepared by freezedrying. The peristomial groove (leading to the mouth) runs along much of the length
of the animal. The cilia in this groove are arranged in groups to form peristomial
membranelles. 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 was
similar but there were differences of note. Cilia arise from the pellicular grooves in
both 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 are
generally less smooth in appearance and they are often bent and are partly attached
to each other or to the pellicle (Figs. 2, 3). The cilia are much smoother and more
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CeU Sci. 4
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A. Boyde and V. C. Barber
'lifelike' in position in unfixed, freeze-dried specimens (Figs. 5, 6). It is not certain
that the cilia are exactly retained in their metachronal position but examination of
parts of the various figures shows that this is a possibility. The other feature to note
is the variable appearance of the pellicular surface. The pellicular ridges are quite
pronounced in some specimens (Figs. 4, 6) whereas others are less obvious and some
very smooth surfaces were produced (Figs. 3, 5). Holes, which are probably freezing
artefacts (they have been seen in other tissues such as muscle) are also seen, and are
generally more obvious in unfixed specimens (Figs. 3, 5). Both the freeze- and airdried OsO4/HgCl2 material (Figs. 4, 10) presented a nodular surface. It is likely that
this result is caused by the large mitochondria and large granules that occur below
the pellicular surface.
Transmission electron-microscopical studies have shown that the membranelles
are arranged in 3 rows of about 12 cilia each, which are separated from the next
group by a peristomial fold, and there is further evidence of this in the arrangement
of the microtubular arrays connected to the basal bodies of each cilium (Daniel &
Mattern, 1965; Finley et al. 1964; Randall, 1957; Yagui & Shigenaka, 1963). These
membranelles are very well seen in fixed specimens (Fig. 7) but are not seen in the
unfixed material, the cilia being separate (Fig. 8).
Spirostomum—air-dried
material
Apart from the OsO4/HgCl2 fixed specimens the different ways of preparing airdried specimens were unsuccessful, the pellicular ridges and grooves being well seen
but no cilia being present. However, specimens fixed by the Parducz method did
have 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 (see
Barber, 1968; Hamlyn-Harris, 1902; Ishikawa, 1924; Young, i960). Essentially, the
statocysts of decapod cephalopods such as Loligo consist of an epithelium, the static
sac, closely applied to the wall of a cartilaginous capsule. In octopods there is a
perilymphatic space between the static sac and the cartilage, but this is absent in
decapods. There are two main sensory regions; the macula, which is a flat plate of
cells overlaid by a crystalline statolith, and the crista, an elongated ridge of cells with
a complicated orientation (see Barber, 1968).
The general appearance of air-dried material can be seen in Fig. 11, which also
shows that a considerable amount of tearing occurs because of the shrinkage during
air-drying. This can be eliminated, or at least largely limited, by freeze-drying. Some
idea of the amount of shrinkage which can occur can be gained from a comparison of
the 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 that
the specimen surface consists of a varnish of dried mucus which obscures details of
interest. Perfusion fixation fixes mucus in situ (Fig. 16). Better results can be obtained
by washing the epithelial surface before fixation, but we have been unable to achieve
reliable removal of mucus with any of a large variety of washing fluids that we have
Freeze-drying of cilia
227
tried. The statoliths were removed in the specimens used in these studies, but some
crystals remain (Figs. 11, 12, 13).
The receptor region in cephalopod statocysts is composed of hair cells and supporting cells. The hair cells bear numerous kinocilia (Figs. 12, 13)—up to 200 per
cell in Octopus (Barber, 1966 a)—and both they and the supporting cells bear
numerous microvilli (Fig. 13). It was not possible to count the number of hair groups
in the air-dried macula, but counts on the freeze-dried material gave a figure of
about 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 airdried material, a much clearer picture was given by the freeze-dried material, where
the 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 such
that the cilia were preserved in their metachronal positions. Our results show that
the Parducz method is much better than the other fixation procedures that we
employed on Spirostomum in fixing cilia in a position which is similar to the one they
would probably occupy when beating in metachronal waves. We would like to
reserve comment on whether this conformation of cilia is 'real', or is a special result
of the Parducz method, but we should like to observe that the Parducz fixation
procedure leaves the cilia in a position which is similar to the position of the cilia in
live, freeze-dried specimens. Incidentally, the Parducz method also leaves the cilia
in a condition where they can be preserved after air-drying.
The fusion of rows of cilia to form the peristomial membranelles is absent in
unfixed specimens. We are inclined to believe that some feature of the quenching and
freeze-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 conventional
electron-microscopical studies which have demonstrated the existence of the membranelles (Daniel & Mattern, 1965; Finley et al. 1964; Yagui & Shigenaka, 1963) also
employed fixed material. The possibility that the fusion of cilia to form membranelles
in fixed material is a fixation artefact, rather than that their separation is a freezing
artefact, cannot be excluded. Grim (1966, 1967), in his transmission electronmicroscopical study of Euplotes using a critical-point freeze-drying procedure on
fixed, partly disrupted specimens, also obtained membranelles.
Freeze-drying techniques again proved their value in the study of the statocyst of
Loligo. The results show that it should be possible to count the exact number of
receptor cells in any ciliated epithelium and a preliminary result of such a count on
the rabbit olfactory mucosa has already been reported (Barber & Boyde, 1968). Such
counts could be correlated with counts of the number of nerve fibres going to, or
leaving, the receptors, for a functional analysis. Another useful possibility is that
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A. Boyde and V. C. Barber
complete receptor areas could be mapped out in other animals as has been done in
Loligo. For example, in Octopus the basal feet borne on the basal bodies of the cilia
point towards the periphery of the macula, and it has been suggested that movement
of the cilia in the direction of their basal feet is excitatory (Barber, 1968). This is
analogous to the situation in the acoustico-vestibular systems of vertebrates (Lowenstein & Wersall, 1959; Lowenstein, Osborne & Wersall, 1964; Flock & Duvall, 1965).
Any conclusion about the squid statocyst must be uncertain without transmission
electron-microscopical information, but if the basal feet do all point towards the
periphery, and movement of cilia in that direction is excitatory, a complete map of
the electrophysiological responses of the cells is now available.
We have found no evidence for or against the existence of a cupula on the crista
of the cephalopod species that we have examined. The structure obviously occupies
the same spatial domain as the mucus-containing fluid of the statocyst, and has only
been reported from light-microscopic studies (as for example by Young, i960, in
Octopus and Young, 1968, in Sepia), where its fragility has been noted. It is probable
that a cupula, if present, would be removed along with the bulk of the mucus in the
preparative procedures that we have used, since these were aimed at washing away
the mucus. We have already noted that perfusion fixation tends to fix the mucus in
situ, and our unpublished scanning electron-microscopical observations have shown
that the cupula is preserved by perfusion of fixative into the semi-circular canals of
an elasmobranch fish.
The asymmetry of the crista in Loligo resembles that of the other cephalopods so
far studied, but no physiological evidence of its function has been reported. However,
electrophysiological recordings from the middle crista nerve supplying the longitudinal portion of the crista in Octopus do show that elements are present that are
unidirectional in response (Maturana & Sperling, 1963).
We would like to thank Professor J. Z. Young for his active assistance and advice. The
visits to the Stazione Zoologies, Naples, Italy, for the collection of cephalopods, were partly
financed by a grant to Professor Young from the U.S. Office of Aerospace Research. We
would like to thank Dr R. Martin for the perfused cephalopod specimens. We should also like
to thank Mr H. Coates, Mr R. Sampson, Mr M. Warrell and Dr K. S. Lester for their help in
constructing the vacuum evaporation unit and cold probe assembly used in these studies, and
Dr M. A. Sleigh and Miss M. Nicholson, who provided us with all our specimens of live
protozoa. 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.).
REFERENCES
BARBER, 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.)
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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 the
analysis 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 the
peristomial membranelle of Spirostomum ambiguum. J. Protozool. 13, 14-27.
FINLEY, H. E., BROWN, C. A. & DANIEL, W. A. (1964). Electron microscopy of the ectoplasm
and infraciliature of Spirostomum ambiguum. J. Protozool. 11, 264-280.
FLOCK, A. & DUVALL, A. J. (1965). The ultrastructure of the kinocilium of the sensory cells
in 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. B
160,
I-I2.
LOWENSTEIN, O. & WERSALL, J. (1959). Functional interpretation of the electron microscopic
structure of the sensory hairs in the cristae of the elasmobranch Raja clavata in terms of
directional sensitivity. Nature, Lond. 184, 1807-1808.
MATURANA, H. R. & SPERLING, S. (1963). Unidirectional response to angular acceleration
recorded from the middle cristal nerve in the statocyst of Octopus. Nature, Lond. 197,
815-816.
MILLONIG, G. (1961). Advantages of a phosphate buffer for OsO 4 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)
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Fig. 1. OsCXj-fixed, freeze-dried Spirostomum. Note the pellicular ridges and
peristomial groove (arrow).
Fig. 2. Higher magnification of a specimen prepared as in Fig. 1. The cilia stand away
from the cell in free space.
Fig. 3. Higher magnification of the part outlined in Fig. 2. Note the somewhat bent
nature of the cilia and the holes in the pellicular surface (arrows), which are probably
a freezing artefact.
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A. Boyde and V. C. Barber
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 cilia
are smoothly curved.
Fig. 5. Unfixed freeze-dried Spirostomum, partly denuded of cilia in preparation. The
specimen 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 the
more irregular surface of the pellicle.
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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 membranelles
are not preserved with this method of preparation.
Fig. 9. An air-dried osmium tetroxide/mercuric chloride fixed specimen of Spirostomum. The pellicular ridges are well seen although the animal has contracted
considerably.
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 method
that we have tried that preserves cilia.
Freeze-drying of cilia
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A. Boyde and V. C. Barber
Fig. 11. The macula of the statocvst of Loligo from a fixed, air-dried specimen. Note
the tearing of the epithelium.
Fig. 12. Higher magnification of the outlined part of Fig. 11 to show the ciliary groups
in more detail. Note the adherent statolith crystals (arrows).
Fig. 13. Higher magnification of part of Fig. 11. Note the individual cilia in the
groups (k) and microvilli (mv).
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A. Boyde and V. C. Barber
Fig. 14. Fixed, freeze-dried specimen of the macula {mac) and crista (cr) in the
statocyst 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. Mucus
obscures some of the details. Each of the ciliary groups is composed of up to 200
kinocilia (arrows).
Freeze-dfying of cilia
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39