Twisted fibrous arrangements in biological materials and cholesteric mesophases

T I S S U E & C E L L 1972 4 (2) 189 217 Published by Longman Group Ltd. Printed in Great Britain

Y. BOULIGAND*

TWISTED FIBROUS A R R A N G E M E N T S IN BIOLOGICAL MATERIALS AND CHOLESTERIC M E S O P H A S E S A B S T R A C T . A c o m p a r i s o n is made between certain fibrous and regularly twisted biological materials and certain ordered liquids c o m m o n l y called 'cholcsteric liquid crystals'. Three examples of twisted arrangements (Arthropod cuticle, Ascidian tunica, DinoIlagellate chromosomes) and typical textures of cholcsteric mesophascs are studied for their optical properties and their defects. These materials are strongly different. Very long polymer chains occur in the organic matrix of skeletal tissues or in chromosomes. On the contrary, in a cholesteric liquid crystal, the molecules are free to move one along the other. However the geometry of such systems is similar. T h e objections to the twisted lnodel arc reviewed and evidence is presented to s u p p o r t a generalized twisted model. A list o f the k n o w n biological cholesteric analogues is given.

number of ordered liquids where the molecules are rather long or could be polymers T w o different kinds of materials will be although the molecules are able to change compared in this paper: on the one hand, we position and are not permanently bound in a shall consider certain fibrous materials which fibrous system. These particular fluids are occur in living organisms and are well known called liquid crystals, mesophases or mesoto biologists and, on the other hand, certain morphic states. Mesophases can be observed ordered liquids mainly studied by physical with certain low-molecular weight pure chemists. substances which pass reversibly from the In general the numerous tissues or cell crystalline state to the fluid state in two or organelles showing a fibrous structure are more steps. They may show birefringence and clearly distinct from liquids even if the mole- still be more or less fluid phases between two cules in the latter are ordered to a certain well defined temperatures tt and tz. Such degree. However, these two different states of mesomorphic states may also occur in a matter may have in common certain geo- solution, the concentration of which varies metrical and optical properties. between two extreme values Ct and C2. More It seems likely that secretions of fibrous complicated mixtures and solutions of monostructures pass through a liquid state, if only or polydisperse macromolecules can also a brief one. The existence of ordered fluids result in the formation of liquid crystals. and possibly ordered liquid secretions could There are two main kinds of mesophases provide an interesting mechanism in morpho(Friedel, 1922 ; see also the recent review by genesis for certain fibrous networks. Chistyakov, 1967). Several biological materials are made of 1. The smectie state where the parallel fibrils arranged according to well defined elongated molecules have their centeis of three-dimensional patterns. These materials gravity arranged on equidistant parallel are often solid and show a strong anisotropy. planes as can be seen from Fig. la. The Similar geometrical organizations occur in a molecules are mobile in the smectic planes. 2. The nematic state, where the parallel * Zoology Dept., E.N.S., 46 rue d ' U l m , Paris 5 °. elongated molecules have their centers of France. gravity arranged at random as in Fig. lb. Manuscript received 17 January 1972 3. The eholesterie state is a variety of the A 189 Introduction

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BOULIGAND

192 and the chromosomes of the Zooxanthellae associated with the sea-anemone Anemmfia sulcata Penn. were sectioned and examined in the phase-contrast and in the electron microscope. The material was fixed, dehydrated and embedded following the routine procedure indicated in the legends to the figures, Liquid crystals of pure substances formed between a microscope slide and the coverslip were studied by means of a polarising microscope. Examples of nematie mesophases are provided with p-azoxyaniso/e (P.A.A.):

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pattern resolvable by electron microscopy and, sometimes, by light microscopy. Each lamina observed in oblique section seems to be composed of fibrils arranged in bowshaped lines (arced or parabolic patterns according to different authors). These bowshaped series are clearlyvisible in thin sections of the Crustacean integument (Fig. 2). In a previous paper (Bouligand, 1965), it has been shown that there are no fibrils which follow the curves and span the distance between two laminae. This appearance is an illusion caused by overlapping planes of fibrils. In each successive layer, the fibril direction is rotated through a small angle about an axis perpendicular to the planes (Fig. lc). The direction of the fibrils rotates through 180" from one lamina to the next. Two oblique sections symmetrical to an axis perpendicular to the laminae give series of

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Results I. Series o f bow-shaped l&es When examined in sections, several biological systems reveal a regular laminated structure. The laminae have a characteristic fibrous

bow-shaped lines with inverted concavity as can be seen from Fig. 3, However, the laminae do not exhibit parabolic patterns in true vertical sections (perpendicular to layers) but alternate bands with fibrils lying parallel to the laminae and to the cutting plane while other fibres run at an angle (Fig. 4 and diagram on Fig. le). The basic properties revealed in various sections are surnmarised in the pyramidal model in Fig. 5. The twisted fibrillar materials are not arranged with the same exactness of the geometrical model. We have considered

Fig. 4. An almost vertical section in the post exuvial layer of a prepuber Carcinus maenas, before calcification of this level. Fibrils in cross section alternate with those in the cutting plane. The bow-shaped appearance is slightly visible, pc, pore-canals; sp, splits determined by the pore-canals. Glutaraldehyde, Osmium, phosphotungstic acid, :,:24,000.

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194

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only a v e r a g e directions o f fibrils in small volumes (the biggest dimensions being only one tenth of the thickness of one lamina at the most). There is a certain degree of disorder defined by the deviation of the fibrils from the average direction in each volume sample. Consequently, the model does not show any fibrils interweaving between horizontal planes at different levels but such vertical connections could occur in the fibrous systems. The twisted fibrous arrangements with b o w - s h a p e d series in thin sections have been observed f r o m m a n y Crustacean a n d insect cuticles, in several other cuticles a n d connective tissue from invertebrates, in vertebrate bones, in certain plant cell walls, in different kinds of extracellular secretions,

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Fig. 6. Vertical section of the crab cuticle at the level of a tubercle. The laminae of the pre- and post-exuvial layers are strongly deformed according to a radial symmetry around the vertical axis. s represents a tangential scction.

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I Fig. 5. Pyramid model. A series of rectangles, where parallel and equidistant straight lines have been drawn, is arranged in the form of a pyramid trunk. in pathological structures, viral tumors, and, m o s t interestingly, in the c h r o m o s o m e s o f Dinoflagellates and n u m e r o u s bacteria (see the list at the end o f the text). This characteristic and striking architecture must have a c o m m o n physical origin in these phyletically a n d functionally very different biological materials. The b o w - s h a p e d series can be seen in true cholesteric mesophases, for instance in mixtures of M.B.B.A. with a small a m o u n t o f C.B. The a r r a n g e m e n t o f molecules is drawn

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TWISTED FIBROUS A R R A N G E M E N T S AND CHOLESTERIC MFSOPHASES on Fig. lc and the obliquity of a section plane introduces the parabolic patterns. Fig. I f gives a scheme of the series in section: the nails are supposed to direct their points towards the observer while the length is proportional to their orthogonal projection on to the plane of the section. When the half helical pitch varies between 10 and 15tL and the thickness of the liquid crystal is of the same order, it is possible directly to see Brownian movements in the ordinary phase-contrast microscope, but the ceaseless movement appears to outline a series of bowshaped arcs which are clearly visible, well defined in position, but it is very difficult to photograph. Series of bow-shaped lines have been also observed under different experimental conditions by Friedel (1922).

2. Spiralized series Where the laminae are distorted in connection with the formation of a tubercle in the body wall of the Crab, a tangential section S of the cuticle (Fig. 6) shows a bow-shaped series in the form of a double spiral (Fig. 9). The origin of this texture is explained in Figs. 7 and 8 where the parallel fibrils now form conical concentric ribbons. In projection, the direction of fibrils along an entire circular ribbon remains constant on to the cutting plane. Since the major refractive indices are parallel to the fibrous directions, alternating dark and bright concentric bands are seen between crossed polaroids (Fig. 10). This change in appearance between double spiral series and bright and dark concentric bands seen in phase contrast and in polarised light, respectively, is also found in thin samples of the cholesteric mesophase when the helical pitch is large (Fig. 13, 14), and the previous explanation also applies to this case. In the periphery of the spirals the laminae may be strongly inclined to the plane of section so that a Maltese cross becomes superimposed on the concentric dark and bright rings seen between the crossed polaroids. 3. Defects ht the twisted arrangement collaborators have indicated some apparent deviations in can, in fact, be explained on the twisted model, for ex-

Neville and recently that insect cuticle the basis of

195

ample preferred orientation of chitin in the tibia of the locust Schistoeerca gregaria and the hind legs of the giant water bug Belostoma. They conclude this partly from the appearance of the parabolic patterns (Belostoma) or from the shape of the pore-canals in cases where the microfibrils could not originally be resolved (Schistocerca). In a later paper the deductions based on porecanals were confirmed by resolving the microfibrils with the aid of potassium permanganate stain (Neville & Luke, 1969b). Neville (1965-67) has also shown that the 'lamellogenesis" can be altered by varying the light and temperature during the period of deposition. During a 'cold night' condition the cuticle appears lamellated and the microfibrils rotate regularly while the day zones are characterized by layers of socalled preferred microfibrillar orientation parallel to the length of the tibia. At certain directions of cutting (plane 3 in Fig. 17) there is strong asymmetry in the parabolic ;f

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Fig. 9 (a) patterns of the lamellate zones (Fig. 15). This effect arises from a preferred orientation of the fibrils along the length of the tibia. The origin of the asymmetry is explained in Fig. 18; asymmetry is difficult to detect in transverse or longitudinal sections (planes 1 and 2 on Fig. 17). For a given thickness, the fibrils then rotate less rapidly in the preferred direction than in the other directions and the twisted arrangement ceases to be regular so that the basic model appears stretched in the preferred direction. The same type of distortion is

196

BOUL1GAND

f o u n d in c h o l e s t e r i c m e s o p h a s e s w h e n a s t r o n g m a g n e t i c field is a p p l i e d parallel to the cholesteric p l a n e (see refs. in K K m a n a n d Friedel, 1969). T h e n u m b e r o f ' l a m i n a e ' in the lamellate z o n e s d e p o s i t e d d u r i n g a n i g h t can c h a n g e f r o m place to p l a c e in t h e s a m e individual, as is s h o w n o n Fig. 16. It s e e m s that p a i r s o f d i s i n c l i n a t i o n lines o c c u r w h e r e t h e l a m i n a e are i n t e r r u p t e d . Such p a i r s h a v e b e e n d e s c r i b e d in r e c e n t p a p e r s o n liquid crystals ( K l ~ m a n et al.) a n d o n e o f the p r e s u m e d a r r a n g e m e n t s is d r a w n o n Fig. 19a, b, T h e axes o f the tubercles o f Carcinus maeaas L. m a y r e p r e s e n t lines o f d i s l o c a t i o n a n d , in this case, t h e t a n g e n t i a l s e c t i o n s d o n o t give rise to a d o u b l e spiral o f b o w - s h a p e d curves but only a single spiral (Fig. 1 I1. Triple spirals m a y also occur. It h a s b e e n s h o w n ( B o u l i g a n d , 1969) that this is c a u s e d by a p a r t i c u l a r a r r a n g e m e n t o f the fibrillar d i r e c t i o n s in the vicinity o f t h e axis o f t h e tubercle, as c a n he seen f r o m Figs. 20, 21, 22. T h e g e o m e t r y o f such tubercles is e x p l a i n e d on Fig. 23a, b, as seen in sections. O n e can see that t h e c o n c e n t r i c b r i g h t a n d d a r k

r i n g s a p p e a r i n g b e t w e e n c r o s s e d p o l a r o i d s will be replaced by spiral b a n d s (Fig. 12). S i m i l a r d i s i n c l i n a t i o n lines h a v e been d e s c r i b e d in t h e cholesteric liquid crystals ( K l d m a n et al.}. I n t h e b o d y wall o f crabs, certain o f t h e s e lines are n o t a s s o c i a t e d with the axis o f a t u b e r c l e a n d can be f o l l o w e d in a s e q u e n c e o f s e c t i o n s t h r o u g h o u t the c o m p l e t e t h i c k n e s s o f the cuticle. 4. CDromosomes in Dim?/tagellates and choh.steric rodh, ts T h e Dinoflagellates are unicellular algae a n d t h e i r c h r o m o s o m e s are c o n d e n s e d a n d visible t h r o u g h o u t t h e c o m p l e t e biological cycle. They show a fibrous structure which appears to be i n t e r m e d i a t e b e t w e e n that o f the bacterial n u c l e u s a n d the c h r o m o s o m e s o f e u k a r y o t e s ( G i e s b r e c h t , 1961). T h e D i n o f l a gellate c h r o m o s o m e s are m a d e o f D N A filaments w h i c h can be clearly resolved in thin section s t a i n e d w i t h uranyl acetate. I n m a t e r i a l fixed in g l u t a r a l d e h y d e , t h e f i b r i l l a r lattice disa p p e a r s after t r e a t m e n t w i t h D N A s e (Puiseux et aLL H i s t o c h e m i c a l tests s h o w an a l m o s t c o m p l e t e a b s e n c e o f histories o r o t h e r basic

Fig. 9. Series of bow-shaped fibrils in tile form of a double spiral. The picture is by a simple spiral of thickening which arises from a microtomy artifact; see the associated diagram Fig, 9 (a}. Alcoholic Bouin's fixative, iron haematoxylin -plus phase-contrast, :. 400. complicated

Fig. I0. The same section between crossed polars (without phase-contrast): the spirals are no longer visible and are replaced by concentric rings, 40{). Fig. I I. Tubercle built around a vertical canal. The bow-shaped series form a simple spiral; the lines of thickening are closed in concentric rings, - 330. Fig. 12. The spiral of Fig. I I is replaced by a spiral of opposite orientation when examined between c r o s s 11icols, > 330, Fig. 13. Mixture of MBBA and CB between a slide and a eoverslip. Phase-contrast. Observe the double spiral arrangenlent of the periodic lmes, ,220. Fig. 14. Preparation in Fig. /3 observed between crossed nicols; spirals are changed into concentric rings, :, 220. Fig. 15. Hind-leg tibia of locust: the cuticle shows an alternation of laminae deposited during the night and unidirectional layers during the day. The obliquity of the cutting plane allows one to scc the asymmetry of the 'crescent' shaped sections of the porecanals; the laminated levels have themselves a preferred orientation which is parallel to the fibrils of the unidirectional layers. The asymmetry of tile bow-shaped patterns is emphasized at top left, - 3400. Fig. 16. Locust cuticle as in the preceding figure. Certain laminae are interrupted in places. The presumed configuration of fibrils at this level is indicated in Fig. 19, × 1700.

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p r o t e i n s a s s o c i a t e d w i t h t h e D N A , as is t h e c a s e in B a c t e r i a (Ris, t 9 6 2 ; D o d g e , 1964). Ultrathin sections of these chromosomes reveal transverse bands of bow-shaped lines. S u c h p i c t u r e s a r e o b s e r v e d in o b l i q u e s e c t i o n s (Fig. 24). I n l o n g i t u d i n a l s e c t i o n , b a n d s o f f i l a m e n t s c u t at r i g h t a n g l e a l t e r n a t e periodically with filanlents which are parallel to t h e p l a n e o f c u t t i n g . I n t r a n s v e r s e o r s l i g h t l y o b l i q u e s e c t i o n s t h e a p p e a r a n c e is t h a t o f a v e r y w i d e b o w - s h a p e d series. All t h e s e p a t t e r n s c o r r e s p o n d to a t w i s t e d arrangement of the DNA filaments. The helical axis is l o n g i t u d i n a l a n d t h e half.,,%-,..

199

MESOPHASES

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Fig. 17. Different orientations of the cutting plane of a tibia of locust. The outer surface of the cuticle is simplified to a cylinder. Sections may be transverse (Pl), longitudinal (p~) or oblique (PaL helical p i t c h lies b e t w e e n 1000 a n d 1500 A . T h e c h r o m o s o m e s a r e m o r e or less e l o n g a t e d , b u t t h e y m a y a l s o be s p h e r i c a l , a n d in t h i s case, t h e d i a m e t e r p e r p e n d i c u l a r to t h e l a m i n a e is t a k e n to r e p r e s e n t t h e l o n g i t u d i n a l axis. S u c h s p h e r i c a l or slightly e l o n g a t e d c h r o m o s o m e s a l s o o c c u r in t h e Z o o x a n t h e l lae a s s o c i a t e d w i t h A n e m o n i a sulcata, a very common sea-anemone. These algae are s y m b i o t i c D i n o f l a g e l l a t e s ( H o v a s s e et al.; Kawaguti). Their chromosomes show an interesting resemblance with the cholesteric d r o p s o r r o d l e t s w h i c h c a n be o b t a i n e d in a state of equilibrium with the isotropic phase, w h e n t h e latter is s l o w l y c o o l e d . E x a m p l e s o f t h e s e d r o p l e t s f l o a t i n g in t h e i s o t r o p i c p h a s e a r e s h o w n o n Fig. 25. T h e relative e l o n g a t i o n o f t h e s e d r o p l e t s o c c u r s e i t h e r by a n i n c r e a s e in t h e t r a n s v e r s e d i a m e t e r or a d e c r e a s e in t h e helical pitch. O t h e r k i n d s o f s p h e r u l i t e s h a v e been described by Robinson (1956-66) and b y S p e n c e r e t a l . (1962) a n d a r e r e l a t e d to very different textures of the mesophase. A three-dimensional reconstruction of the

Fig. 18. Model for the interpretation of the nonsymmetric bow-shaped patterns in the cuticle. (a) If a twisted model is stretched in a direction p lying parallel to the plane L of the laminae, an oblique section to p and L gives a pattern detailed on b. At the level s parallel to p, the rotation of filaments is slow. At the level r, perpendicular to p, the rotation is more rapid.

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BOULIGAND

200

been crystallized from isotropic solutions of polyethylene derivatives. Long polymer chains can pass through the interfacial zone, from the mesomorphic phase to the isotropic liquid, as can be seen from the model illustrated in Fig. 27. This rather surprising comparison between chromosomes and certain cholesteric arrangements in plastic materials underlines the possibility that very long asymmetrical polymer chains can associate in this typical fashion.

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Fig. 20. When filaments are arranged in a layer according to homofocaI parabolae, the direction of filaments in a circular ribbon turns of i80" on the whole ring. outer shape of the chromosomes of Zooxanthellae (Fig. 26) has been p u n i s h e d (Bouligand eta[. 1969) and underlines the relation with the droplets in the cholesteric mesophase floating in the isotropic liquid. The extremities of the rodlets show regular double-spirals ( Fig. 25) which occur also in the chromosomes. It is highly probable that some long filaments of D N A extend a certain distalace into the nuclear sap and could form loops~ as shown in Fig. 26. A utoradiographic studies (Babillot, 1970) on Dinoflagellates have shown that tritiated uridine is incorporated in the nucleoplasm and not in the chromosome bodies themselves. These loops could therefore be involved in the R N A synthesis. Rodlets and spherulites with a cholesteric periodicity have ,i

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Fig. 21. Arrangement of concentric conic ribbons in the thickness of a section: the axis of the parabolae turns regularly from one ribbon to the next.

This paragraph is necessary in order to understand certain artifacts which may apply in thin sections of twisted fibrous materials. The classical cholesteric mesophases have never been subjected to microtomy. However, the thin mesophases between microscope slide and coverslip may show distortions which resemble certain microtomy artifacts (paper in preparation). W h e n a twisted fibrous material is sectioned, the microtome knife meets the filaments in alternate different directions. The block and the knife have different elastic characteristics and are submitted to periodic tensions. Lines of compression or thickening can arise from such a system. The bowshaped series are often complicated by reguIar bands of higher optical density. Both mechanical and optical artifacts may therefore be involved in the patterns of the dark periodical bands. Certain simple cases are explained in Fig. 28. The filaments shown in Fig. 28a are sectioned and slightly turned up at p, but are only separated at level q. The successions of steps on the two sides of a section may be displaced to a certain degree, depending on the mean thickness and on the incidence of the laminae in the cutting plane. The bands may also change in width and in degree of overlap, as is seen in Figs. 29 and 28, b e. In the case of Arthropod cuticles, such a view of the sections has often been interpretated in terms of alternating layers, either marked by distinct chitin and protein composition (Bouhniol etal., 1964; Horridge, 1969) or more simply made of denser or less dense cuticular material (Richards, 1951). Comparable interpretations have been proposed by many authors working on different kinds of twisted fibrous materials (Boyde et al., 1969; Chafe, 1970), but in all the cases,

TWISTED FIBROUS ARRANGEMENTS

AND CHOLESTERIC

MESOPHASES

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Fig. 22. The projection on the cutting plane of the directions of filaments lying in the successive concentric ribbons leads to a simple spiral of bow-shaped lines. One of the concentric ribbons is emphasized. the structure is m o s t likely h o m o g e n o u s ; the claimed chemical or physical a l t e r n a t i o n does n o t exist a n d the lamellate a p p e a r a n c e arises f r o m section artifacts in a twisted arrangement. T h e long corneal cones of the c o m p o u n d eye of Photm'is are l a m i n a t e d in a series o f concentric paraboloids. In transverse section, the l a m i n a t i o n s form a single spiral (Fig. 30) b u t the axis o f this spiral a r r a n g e m e n t seems to h a v e never been observed in sublongitudinal section (Horridge, 1969). T h e particular aspect of transverse sections can be interpreted on the basis of three a s s u m p t i o n s :

(1) T h e l a m i n a e are of cuticular n a t u r e a n d the fibrils are a r r a n g e d a c c o r d i n g to the twisted model. (2) T h e fibrils are n o t well resolvable in t h i n sections (like in the Locust tibia cuticle).* (3) T h e m a x i m u m opacity follows either the locus of p o i n t s where the filaments lie parallel to the section plane, or the middle line of the t h i c k e n e d regions where the One can resolve fibrils, in certain cases (Neville et al., 1969) using a method due to Thi6ry (ar. Microscopie, 4, 165, 1965).

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Fig. 23. (a) Two families F1 and F2 of homofocal parabolae. The second family (dotted lines) cut the first under a constant angle c~. Fz is said to be isoclinal to the F1 family. The axis of the first and the second families make an angle 2a. This property is explained on Fig. b. (b) The focus F is common for the two families; the two axes a 1 and az are separated by an angle 2c~. Consider any point M and the parallel MH1 to Fal. MH~ is supposed to be equal to MF and then the straight line D~ perpendicular to MH 1 is the directrice of the parabola P~ of the first family passing by M. The directrice D~ is defined in the same way. The tangents MT~ and MT~ in M are the bissectrices of the angles FMH 1 and FMHz respectively. The angle of M T 1 and MTz is then equal to ~. filaments are in position to be t u r n e d up by the knife edge. A twisted model a r r a n g e d on concentric p a r a b o l o i d s gives in transverse section two series of arcs as has been s h o w n previously in Figs. 6-8. The b o u n d a r i e s of the b o w - s h a p e d series f o r m a double spiral c o r r e s p o n d i n g possibly to a weak m a x i m u m of density. Indeed, in the case of filaments which are h o m o g e n e o u s l y distributed, but n o t closely p a c k e d together, the electron opacity is weaker for a cross-section t h a n for a longitudinal one. The relative o r i e n t a t i o n of the twisted a r r a n g e m e n t is s h o w n in different cubic blocks (Fig. 31) and, in each one, two a r r o w s indicate the b o u n d a r y between the regions where the filaments are slightly t u r n e d up (p) or n o t (q) by the knife edge. Stippled zones in the double spiral of Fig. 31 represent the (p) regions. T h e strongest effect of compression a n d thickening occurs along the middle line of these regions which is a simple spiral. The superposition of these simple a n d double spirals explains completely the regular irnages observed in transverse section. Fig. 30 has been d r a w n from a m i c r o g r a p h of a p s e u d o c o n e (cover of Science, N ° 3845) a n d two systems of d a r k spiral lines are indicated with a simple or

double a r r o w respectively. Similar images h a v e been analysed in the crab cuticle where c o m p a r a b l e a r r a n g e m e n t s of l a m i n a e in concentric p a r a b o l o i d s occur in the dorsal body-wall, for instance (Fig. 9). T h e simple spiral a n d the d o u b l e b o w - s h a p e d series are clearlyvisible at the same time (see Fig. 9 bis). T h e l a m i n a t i o n has been assumed to arise f r o m the protein c o n c e n t r a t i o n a n d therefore the refractive index of the material (Horridge, 1969). T h e chemical c o m p o s i t i o n is however most likely h o m o g e n o u s . The material is certainly birefringent as are o t h e r insect cuticles in vertical section. T h e index ellipsoid is everywhere the same, but its spatial distribution is twisted according to the helical pitch of the laminae. It is well k n o w n that strong reflections can occur o n such materials which are optical analogues of cholesteric liquid crystals (Gaubert, 1924; R o b i n s o n , 1966; Bouligand, 1969 ; Neville, 1969 : et al.) and, thus, it is clear that the p a r a b o l o i d s can b e n d rays towards the optical axis within the cones. The question of the alternation of layers of different physical or chemical n a t u r e needs further investigation in a n u m b e r o f biological materials and, for instance, the

Fig. 24. Thin section of a Dinoflagellate nucleus (Xanthella of Anemonia sulcata). The c h r o m o s o m e s show a twisted fibrous a r r a n g e m e n t ; in oblique section, one recognizes the bow-shaped series (~j and in longitudinal section, the alternation of filaments lying in the cutting plane and those cut transversely (fl). The bodies of the c h r o m o s o m e s are separated from the nucleoplasm (n) by a white halo which is most likely caused by a shrinkage of the D N A filaments. This halo is crossed by m a n y thin filaments extending towards the nucleoplasm, ~, 40,000. Fig. 25. Suspension o f cholesteric droplets in the isotropic liquid (mixture of MBBA, CB and toluene). Analyser only. A dislocation is visible in the droplet at the top of the figure; such arrangements occur also in the c h r o m o s o m e s of the Dinoflagellates, • 800.

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F i g . 26. T h r c e - d i m e n s i o n a l reconstruction of the external shape of a Xanthclla chromosome a n d its r e l a t i o n s w i t h t h e n u c l e a r s a p . T h e f i l a m e n t s e x t e n d i n g t o w a r d s the nucleoplasm probably form a number of loops.

alternation of thin continuous lamellae of c h i t i n a n d r e s i l i n in c e r t a i n r u b b e r - l i k e i n s e c t c u t i c l e s s e e m s a l s o to be q u e s t i o n a b l e ( N e v i l l e , 1965; W e i s - F o g h , 1 9 6 0 - 7 0 ) . Discussion

1. t t l S T O R I C A L RESUM[~ The twisted model for certain fibrous biol o g i c a l m a t e r i a l s w a s p r o p o s e d in 1924 b y

F i g . 27. T h e o r e t i c a l s t r u c t u r e o f b i r e f r i n g e n t r o d s in the isotropic liquid during the crystallisation of polymers in certain plastics. A small percentage of long chain molecules passes through the interface between the birefringent phase and the isotropic liquid. This picture shows an interesting analogy with the equilibrium between the chrornosomes and the nucleoplasm.

Schmidt who studied the integument of the crab Cancer pagurus and the mantle of the tunicate Halocynthia papillosa. His conception of the fibrous arrangement has been p r o d u c e d in t h e c l a s s i c a l w o r k s o f F r e y W y s s l i n g (1951) a n d P i c k e n (1960). T h e i d e a o f S c h m i d t is d e r i v e d f r o m a p o l a r i z i n g microscope analysis. The fibrous components can introduce a strong form birefringence a n d all v e r t i c a l s e c t i o n s o r i e n t e d a t 45 ° to t h e c r o s s e d n i c o l s s h o w a l t e r n a t i n g b r i g h t a n d d a r k lines. S c h m i d t w o r k e d s o m e years after his predecessors Biedermann (1903, 1917) a n d H a s s (1916), w h o h a v e put forward a 'plywood' pattern for this material. S c h m i d t (1924) p o i n t e d o u t t h a t v e r t i c a l s e c t i o n s a t 4 5 ' t o t h e t w o c r o s s - w i s e fibrill a t i o n s w o u l d s h o w t h e s a m e b e h a v i o u r in t h e p o l a r i z i n g m i c r o s c o p e . T h e s i m i l a r i t y o f all the vertical sections suggested strongly a helical model. Schmidt did not realize that his model c o u l d l e a d to a n e x p l a n a t i o n o f t h e b o w shaped appearance of the filaments and he s u p p o s e d o n t h e c o n t r a r y t h a t c e r t a i n fibrils were not horizontal but connected different levels, s t r e n g t h e n i n g t h e m a t e r i a l .

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Fig. 28. D i a g r a m o f the m e c h a n i s m o f m i c r o t o m y in a twisted material (a) the knife edge K alternatively either passes between the filaments or cut t h e m . T h e cut ends o f the filaments are slightly turned up. This m e c h a n i s m leads to steps which can be shifted to a certain degree between both sides of the section. A t the microscopic examination, the section in b gives dark b a n d s alternating with clear ones (c). T h e b o w - s h a p e d a r r a n g e m e n t is indicated at the m e a n horizontal level, d, e: in certain cases, the d a r k bands m a y overlap and this leads to still d a r k e r ones.

Later, Drach (1939, 1953) studied the cuticle of crabs and made certain objections to Schmidt's model. (1) The reality of the laminae is shown by the fact that they are mechanically separable (Hass, 1916; Dennell, 1960). (2) The visible periodicity of the laminae in section is due to alternating levels: intermediary zones (I, dark, narrow, with horizontal filaments); fundamental zones (F, light, larger, with arced filaments, Drach 1939 and Dennell 1960). (3) A piece of mineralized cuticle can be limited by surfaces of fracture forming a rectangular dihedron. At the vertical edge, the F- and 1-bands are not interrupted (Fig. 33). On the contrary, Schmidt's model would predict an exchange between I- and F-bands. Drach (1953) tried to reconcile the polarizing microscope data of Schmidt and the old conception of well defined and separated laminae. In this model (Fig. 32a) l-levels are thin and contain horizontal filaments

only. The F- ones are thicker and composed of arced filaments lying in parallel oblique planes and linking tangentially both contiguous I-bands. It is not easy to criticize such a conception, because a very small angle e of obliquity with the horizontal plane is enough to make the Drach's model as near as possible to that of Schmidt (Fig. 32d). In contrast to the models by Drach (Fig. 32a) and Locke (Fig. 32b) 1 have shown (Bouligand, 1965) that arced filaments are not necessary to explain the images. The twisted disposition of filaments seems to be the only factor responsible for the bowshaped appearance of the series. The arguments which have been put forward against the twisted model are reviewed below. 2. CRITICAL STUDY OF THE OBJECTIONS TO THE TWISTED MODEL (A) Delam#lation ; l and F bands. Horizontal cleavage is frequent in the mineralized

206

BOULIGAND

Fig. 29. Alternating dark and light bands in decalcified cuticle of Carcinus maenas. At the top of the figure, a certain overlap of the dark bands lead to darker ones as can be understood from Fig. 28. This figure does not show any physical or chemical difference between the bands but only three orders of thickness which are artifacts of microtomy. The relative thickness of the section does not allow a good observation of the bow-shaped series (The superimposed concentric lines correspond to an alternation of density of the haematoxylin staining in the region of radial growth of the Calcite which has been previously removed from the blockl. Bouin's lixative, iron haematoxylin, phase-contrast.

cuticle of Carcinus maenas but occurs preferentially at the limit of the pre- a n d post-exuvial layers. T h e fracture surfaces are i n d e p e n d e n t of the l a m i n a e in most parts of the body. However, exceptions have been observed in certain zones of the a b d o m e n a n d in the podomeres. T h e I-levels in section do not necessarily show the m a x i m u m optical density. T h e d a r k b a n d s a n d the b o w - s h a p e d series have an equal periodicity b u t m a y be out of phase. (See for instance the spirals of the Fig. 9.) T h e c o n t i n u i t y of I - b a n d s a n d F - b a n d s across the vertical edge of a rectangle d i h e d r o n (Fig. 33) can be i n t e r p r e t e d in terms of twisted model a n d translucence on the t h i n n e r p e r i p h e r y of the block. T h e calcite a n d the fibrils have different optical indices a n d s t r o n g reflections occur on filaments. H o r i zontal light is reflected u p or d o w n a c c o r d i n g

to the levels I or F (Fig. 24d). W i t h a diametrally opposite light-source, I-bands t u r n F a n d vice versa, as can be seen from small particles, mechanically attached to the surface of the d i h e d r o n (Fig. 33a, b; Fig. 34a, b, c). This o b s e r v a t i o n m a d e o n the cuticle of Carcinus maenas has been r e p r o d u c e d o n a small rectangle d i h e d r o n with vertical edge cut in the tunica of Halocynthia papillosa. T h u s the objections raised against the twisted m o d e l seem to afford o n the contrary new a r g u m e n t s in its favour, especially in the case of the two materials e x a m i n e d by Schmidt (1924). (B) Generalized twisted model, The first description of the model (Fig. 5) applies to a flat region of the cuticle with straight filaments at each level. Consider a set of parallel surfaces a n d the family of n o r m a l

TWISTED FIBROUS ARRANGEMENTS curves (Fig. 35). Between two of these surfaces, St a n d S~,, the n o r m a l m e a s u r e d distance is a c o n s t a n t Ae. These surfaces will be called i s o c h r o n a l a n d are supposed to represent the level secreted at a time t. T h e complexity of shape is well k n o w n for the a r t h r o p o d cuticle a n d other twisted fibrous materials. The fibrils of a surface S separated by a large distance are not necessarily parallel, the surfaces being n o t flat. We have to consider a family of curves C1 o n St, C2 on S~ e t c . . .; if a n o r m a l line meets S~ in M~ a n d Su in M~, the angle A0 between the tangents M T ~ to Ca a n d M2T.., to C.~ is c o n s t a n t for two chosen S~ a n d S~. The C~ curves are said to be fisoclinal' to C~. W h e n the thickness of the laminae is constant, ~ 0 is p r o p o r t i o n a l to Ae. However, the helical pitch can vary in a c o n t i n u o u s m a n n e r according to the level or the place in the surface S. A simple case of this generalized model is given w h e n S are planes, but C are n o t straight lines, the pitch being constant. T h e l a m i n a e cease to be parallel to the secretion levels S as it will be seen f r o m Fig. 36. M a n y examples of such a slight obliqueness of laminae o n the cuticular direction have been recorded in crustacean cuticle. This m e a n s p r o b a b l y that layering m a y occur earlier in certain regions, the total n u m b e r of l a m i n a e a n d the whole thickness are highly variable. T h e speed of layering could vary locally a n d the secretion could start in determined zones a n d spread a r o u n d , which would lead to n o n h o r i z o n t a l laminae. W h e n a piece of cuticle is fixed in the course of the fibril deposition, one c a n detect in certain restricted regions a slight obliqueness of the laminae to the last isochronal surface (i.e. the inner surface of the u n c o m p l e t e d cuticle). This is a strong a r g u m e n t for the D r a c h model in certain zones. Certain l a m i n a e are i n t e r r u p t e d here a n d there at the b o u n d a r y between the l a m i n a t e d levels a n d the unidirectional levels of the tibial cuticle of locusts. These b o u n daries are u n d e r control of the light conditions a n d are p r o b a b l y isochronal surfaces, M a n y examples of obliquity between l a m i n a e of different levels can be f o u n d in this material. D r a c h ' s model, with a very low angle ~,. of the planes of arced lines on the h o r i z o n t a l is a particular case of the generalized twisted fibrillar a r r a n g e m e n t (Fig. 32d). Suppose

AND CHOLESTERIC

MESOPHASES

207

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Fig. 30. Drawing of a micrograph published on the cover of Sciettce n ° 3845. Tangential section of the compound eye of a firefly. A cuticular pseudocone shows a single dense spiral which is locally divided into two. Simple or double arrows refer to the interpretation on the next figure and underline the single and double spirals interfering in this micrograph. same shape). There are h o w e v e r b o w - s h a p e d lines of equation), - Y0 + loge I cos x I, x a n d y m e a s u r e d with the same unity, which are equal to their own isoclinals, This particular D r a c h ' s model c a n give arced series with opposite concavity, w h e n sectioned in certain particular directions. In this case, the first factor of the b o w - s h a p e d a p p e a r a n c e in oblique section r e m a i n s the twisted disposition of filaments a n d n o t their widely a r c e d shape. T h e distinction between D r a c h ' s model a n d the twisted model w o u l d have n o great physical significance w h e n ~ is n o t a b l y inferior to the deviation of filaments in small volumes a r o u n d their average direction. This deviation o n the c o n t r a r y m a y be estimated a n d is often i m p o r t a n t as can be seen o n u l t r a t h i n sections. N o t e t h a t D r a c h ' s configuration would n o t b e stable in a true cholesteric liquid crystal. The occurence o f such a n arrangem e n t in certain restricted zones would result f r o m a fast h a r d e n i n g (a p s e u d o m o r p h o s i s ) if the material passes t h r o u g h a genuine

BOULIGAND

208

km

Fig. 31. Idealized double spiralized section of a tubercle in Carcinus maenas or a pseudocone in a firefly. The limits of the bow-shaped series are in the form of a double spiral. The blocks indicate the relative orientation of the twisted arrangements in ten radial sectors and explain the orientation of the concavities in the complete figure. In each block, one can distinguish on the frontal face two zones where the fibrils are either turned up (p) or not (qt by the relative motion of the knife edge indicated by the arrow kin. The stippled zones represent the p regions. The middle line of this latter corresponds to a maximum of thickening or compression of the section and is in the form of a single spiral. See Fig. 9 and 9 bis. The same kind of study leads to an interpretation of the concentric rings of thickening in horizontal sections when the tubercle gives a series of bow-shaped lines in the form of a single spiral (see Fig. 11).

m e s o p h a s e at t h e time o f the secretion. T h e surfaces S of the generalized m o d e l do n o t necessarily exist w h e n the twist axis are n o t parallel in each p o i n t to a g r a d i e n t field. S u c h s y s t e m s c a n occur in t h e fibrillar twisted m a t e r i a l s b u t this result does n o t c h a n g e the e x p l a n a t i o n o f the o b l i q u e n e s s o f certain l a m i n a e . (c) Recent objections to the twisted model. B o w - s h a p e d series h a v e b e e n o b s e r v e d by R u d a l l (1969) by n e g a t i v e s t a i n i n g or s h a d o w -

ing o f t h i n layers, stripped to the i n n e r s u r f a c e o f the cuticle o f locusts, kept u n d e r c o n t i n u o u s n i g h t c o n d i t i o n s . (i.e. refering to Neville's e x p e r i m e n t s , with c o m p l e t e l y l a m e l l a t e endocuticle). T h e d e t a c h e d layer could be t h e s u b c u t i c l e defined b y E. L. S c h m i d t (1956). It is p o s s i b l y a n e x a m p l e o f D r a c h ' s m o d e l w i t h low angle. T h i s p o i n t o f view is n o t c o n t r a d i c t o r y to t h e c o n c l u s i o n s of Neville d i s c u s s e d above. S u c h an a r r a n g e m e n t closed to t h a t o f the

TWISTED

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.

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.

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CHOLESTERIC

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<-,~ / s " ~-

-7 H

lJ....s

~.:':'! ,".,I' il~.%,,~ .,'ii

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Fig. 32. Different models of Drach. (a) Original where c~is not far from 45". (b) The conception of Locke: o~ 90 °. (c) The Drach's model applicd to the tunica of H a l o c y n t h i a by Neville. (d) Drach's model when 0~is very low. The thickness of the laminae is small as compared to the width of the arced series. Such a system introduces a strong twist between the filaments and approaches Schmidt's model.

twisted m o d e l leads to the 'crescent' shapes of p o r e - c a n a l sections. It seems h o w e v e r t h a t the a s s u m p t i o n s of R u d a l l need further investigation. (1) T h e r e is n o clear evidence t h a t the cleavage surface a n d the filaments are perfectly parallel. D i s p e r s i o n of filaments a r o u n d t h e i r average direction allows a certain interweaving. T h e s h a d o w i n g o f fibrous layers shows filaments which are i n t e r r u p t e d here a n d there by cleavage. A n u m b e r o f irregularities in the b o w - s h a p e d

series p u b l i s h e d b y R u d a l l (1969) could arise f r o m this m e c h a n i s m . (2) As has been p o i n t e d out by Rudall, ' t h e separation is subject to m u c h variation, the concavity o f the arcs c a n show different o r i e n t a t i o n s a n d there are regions of considerable swirling or whorling'. T h i s description is typical o f a tengential section in a true, b u t s o m e w h a t distorted, twisted model. I n the low angle hypothesis for D r a c h ' s model, the oblique planes would h a v e then n o preferred o r i e n t a t i o n on a large scale,

210

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Q

>

Fig. 33. Examination of a dihedron of calcified cuticle of Carcinus maenas. Small particles mechanically attached to thc surface of the fracture and indicated by arrows are seen on clear or dark bands according to that the light comes from bottom !eft (a) or top right (bL This shows clearly the relative character of the 1- and F-bands, ;. 340.

(3) M a n y dislocation surfaces can lead to preferential level o f cleavage and certain laminae will be sectioned obliquely. This case is not rare in the endocuticle and then at the subcuticular level. Neville (1967) asked the question as to whether the twisted model did really fit the case of the tunica of the Ascidia Halocynthia papillosa, and this for the following reasons: (1) "The alignment of parabolae on consecutive faces of the pyramid model (Fig. 5) changes by half a register. This does not occur in tunicate lamellae.' (2) 'The direction o f parabolae in the model does not change from face to face, whereas in the tunicate example, as can be worked out f r o m Fig. 32c, the sequence is left, left, right, right.'

W e have cut small pyramidal trunks in the

Haloo'nthia tunica. The oblique faces show very clearly the lamellate appearance, but the arced lines are not resolvable with the binocular microscope. After dehydration, clearing in xylol and m o u n t i n g in balsam, the fibrils are clearly visible with the phasecontrast microscope and the parabolic patterns arise from the optical sections and are not due to the obliquely sectioned sides of the pyramid (see Fig. 37). The observations of Neville can be interpreted on this basis and are not in contradiction with the twisted model. W h e n a piece o f curved tunica is cut tangentially for histology, there is a clear inversion between the concavities of the bowshaped lines in diametrally opposite regions,

Fig. 34. Theoretical scheme related to the Fig. 33. (al A rectangle dihedron immersed in water, and lighted either by the source $1 or $2, is examined through a microscope M, (b, c). The image depends on the orientation of the light: the attached particles p are seen on clear bands with the light on the left and on the dark ones with the light on the right. (dl The light is reflected up or down according to the direction of filaments, the optical index of which is markedly different from that of Calcite. This effect is inverted obviously with the orientation of light.

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212

BOULIGAND

This fact does not fit the Drach's model with a strong obliquity of the arced filaments on the horizontal planes as is required by Neville's assumption.

/,,,,,,,

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Fig. 35, Principle of the generalized twisted model (see the textl. 3. TWISTED FIBROUS S T R U C T U R E S AND CHOLESTERIC MESOPHASES

The helical character of cholesteric mesophases has been demonstrated by Mauguin (1911) and Ftiedel (1922) who pointed out the close relation of this special mesomorphic state with the nematic one. A more complete description was given later by de Vries (1951) on theoretical grounds and by Robinson (1956-1966) who prepared beautiful cholesteric phases with synthetic polypeptides. The basic arguments of Robinson

~/S$..'..~;<,.,//lllll,

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on the cholesteric structure resemble closely those of Schmidt (1924). The cholesteric liquid crystals show remarkable optical properties including strong rotatory power and selective reflection of circularly polarized light in a narrow region of wave-lengths. These phenomena were known for a long time in certain insect cuticles and the relation with cholesteric phases had been emphasized by Michelson, Gaubert, Mathieu and Robinson (see Robinson, 1966). A twisted arrangement of the chitin-protein network has been found in the cuticle of the beetle Cetonia aurata, at the levels which reflect circularly polarized light and show a strong rotary power (Boufigand, 1969). On the contrary, in Chrysocarabus, another beetle reflecting the same metallic colouring, without circularly polarized light, the outer part of the cuticle (of a probable epicuticular nature) is made of alternating dark and light bands in section, with no sign of any twisted arrangement (Bouligand, 1969). Another pattern without any twisted element has been described in certain Cetoniinae (g. Heterorrhina) which reflect a metallic but not circularly polarized light (Neville and Caveney, 1969). Summary and Conclusions The twisted biological materials and the ¢holesteric liquid crystals show many geometrical and optical properties in common. The two systems show similar dislocations. The microscopic examination of the twisted fibrous arrangements is often complicated by artifacts which are strongly misleading for histochemical interpretations. The first conception of the twisted model in certain biological fibrous materials with overlapping planes of straight parallel filaments, had to be adapted to the morphological complexity. A generalized twisted model with overlapping surfaces containing slightly curved filaments gives a more exact representation. Variations of the helical pitch and certain preferred orientation must also be introduced. This new point of view allows an interpretation of certain slight obliquity recorded between the laminae and the deposition surface of the fibrous material. Drach's model with an extremely low angle of obliquity of the bow-shaped filaments could cccur in certain restricted

TWISTED FIBROUS ARRANGEMENTS

AND CHOEESTERIC

MESOPHASES

213

.~,,," - , :"-L-" ~ "2-~ L'-..:~ ~:5-~-

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'

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:

J

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•

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Fig. 37. Explanation of the Neville's observations on the Ascidian tunica. A trunk of pyramid is cut in the tunica, the two parallel faces being parallel to the laminae. Such a system is examined obliquely throngh the phase microscope with a small focusing latitude: the observed bow-shaped series correspond only to an optical section. When the focus is changed (three different levels are considered on the figure) the arced patterns follow the principal directions of the pyramid (arrowsl and does not change half a register at the level of thc oblique edges.

zones of twisted materials. This a r r a n g e m e n t is a particular case of the generalized twisted model. However, in this extreme hypothesis with very large arced filaments, it is n o t generally the shape of filaments themselves which is responsible for the arced aspect of laminae in section but the twisted arrangem e n t w h i c h still occur in this r a t h e r particular model. D r a c h ' s model in its original form a n d Locke's model i m p l y periodic m o t i o n s of the secretory cells in order to explain the arced shape of filaments, in the case of skeletal tissues. The histochemical misinterpretations have led to the a s s u m p t i o n of a secretion r h y t h m related to the alternate layers o f different natures. T h e twisted model removes these difficulties a n d affords a n attractive m e c h a n i s m for the l a m i n a e genesis, which seems closely related to the growth of cholesteric mesophases.

Tentative list of the twisted fibrous materials:

I--Cytoplasmic inclusions. Proteins in pancreatic acinar cells of the mouse; B a n n a s c h , 1966. Inclusions in s y m p a t h e t i c n e u r o n s : Se~te, 1969. H a e m o g l o b i n in sickle-cell a n a e m i a erythrocytes: D6bler, 1968. Kinetoplastic D N A of Trypanosonta crttzi after a t r e a t m e n t with e t h i d i u m b r o m i d e ; D e l a i n a n d Riou, 1969. Viruses in cells o f plants a n d Insects hosts; Shikata a n d coll., 1969, See also: W i l s o n etal., 1970.

11--Skeletal structures Central capsule of certain R a d i o l a r i a n s P h a e o d a r i a ; L6cher et Cachon, 1970. Cuticle of Aurelia attrita, M e d u s a e S c y p h o s t o m a e ; C h a p m a n , 1968.

BOULIGAND

214 Cuticle of Philodina roseola Rotifer; Dickson and Mercer, 1967. Cuticle of Nematoda (Mermis); Lee, 1970. Cuticle of Arthropods: Crustaceans; Bouligand, 1965 a, b. Insects; Locke, 1964; Neville, 1969. Bouiigand, 1969. Myriapods; Silvestri, 1901. Arachnids: Barth, 1970. Tunica of Halocynthia papillosa (Tunicate) Bouligand. Compact bone of Vertebrates; Frank et a/., 1955; Bouligand. Ill--Connective tissues of certain Invertebrates. Hm,elockia hwrmi~" and Thyone (Holothuro[d, Echinoderm); Gross, Piez, 1960; Bouligand. Discocoelides hmgi (Turbellaria, Polyclad): Pedersen, 1966. IV-~Laminated levels" in membranes of certain attimal eggs. Diopatra cupraea, Polychaete; Anderson and Huebner, 1968. Two,mastix, Crustacean Phyllopoda; Bouligand. drtemia salina, Crustacean Phyllopoda; Morris and Afzelius, 1967. Me[anophts, Acheta; Orthoptera; Slifer and Sekhon, 1963: Furneaux el al., 1969. Qvnolebias, Teleost; Sterba and Mfiller, 1962.

V--Cell walls Collenchyme in several plants; Chafe, 1970. Oat coleoptile; O'Brien and Thimann, 1966. Endogone spores; Mosse, 1970. V1--Bacterial mwleus and D&qflage[[ate Chromoxon?es. Bacillas megaterium; Giesbrecht, 1960. Bacillus subtilis; Ryter, 1963. AmphM#tiunt; Grel[ and coll., 1957. de Haller and coll., 1964. See also Grund, 1965.

VI l--Polyethyh, nes; Keller and O'Connor, 1958.

Acknowledgements

I wish to thank Prof. T. Weis-Fogh, Drs. C. A. Neville, B. L. Gupta and P. Cladis for their comments on the manuscript and for providing encouragement.

Added in Proof

Interesting bow-shaped arrangements have been observed recently in tissues of different hosts of viruses Alfalfa mosaic virus, Hull et al., Virology 42, 753 (1970) Virose of the garden pea, Rassel, C. R. Acad. Sci. Paris', D, 274, 2871 (1970).

TWISTED

FIBROUS

ARRANGEMENTS

AND

CHOLESTERIC

MESOPHASES

215

References ANDERSON, E. and HUEBNER, E. 1968. Development of the Oocyte and its accessory Cells of tile Polychaete Diopatra eaprea (Bosc.) J. Morph., 126, 163-198. BAB~Lt,OT, C. 1970. Etude de I'incorporation d'uridine triti~e dans Ie noyau d'Amphidinittm earteri (Dinoflagelld). C. r. bebd. &;one. ,4cad. Sei., Paris, D, 271, 828 831. BANNASCI~, P, I966. Htillenlosc Cytoplasmainclusionen und ihre Beziehong zur Sekretbildung im exokrinen Pankreas der Maus J, Ultrastrllet, Res,, 15, 528 542. BART~t, E. G. 1966. Die Feinstruktur des Spinnemnteguments. 1. Die cuticula des Laufteins adulter hfiutungsferner Tiere. Z. Zell/orsch mikrosk. Anat., 97, 137-159. 1969. BARTH, F. G. 1970. Die Feinstruktur des Spinneninteguments. II. Die rafimliche Anordnung der Mikrofasern in der lamellierten Cuticula und ihre Beziehung zur Gestalt der Porenkanfile (Capiennius salei Keys, adult hfutungsfern. Tarsus), Z, Zel!fbrsch., mikrosk. Anat., 104, 87-106. BERTH~S, J. F. and D~]BLER, J. 1969. Reversible and irreversible sickling: a distinction by electron microscopy. Blood, 33, 884 898. BIEDERMANN,W. 1913.1 n Winterstein, Handbuch der ~,ergleichenden Physiologic. 3, Physiologic d er StLitz- un d Skelettsubstanzen, 814- 912. BIEDERMANN, W. 1917. Sekretion und Sekrete. P!Tiigers Arch. ges Physiol., 167. BOULIGAND, Y. 1965a. Sur uric architecture torsadde rdpandne dons de nombreuses cuticules d'Arthropodes. C.r. hebd. &;one. Aead. Sci., Paris, 261, 3665--.3668. BOULmANI), Y. I965 b. Sur une disposition fibrillaire torsadde commune'a plusieurs structures biologiques. C, r. hebd. S&tne., Acad. Sei., Paris, 261, 4864 4867. BOULmAND, Y. 1967. Comparaison de certains mat6riels biologiques "a la structure des cristaux liquides 'cholestdriques'. J. Mieroscopie., 6, 41 a. BOULIGAND, Y. 1967. kes soles et les cellules associdcs chez deux Anndlides polychbtes. ~,tade en microscopie photonique h contraste de phase et en microscopie 61ectronique. Z. Zellforsch. mikrosl<. Anat., 79, 332-363. BOULIGAND, Y. 1968. Sur une eat6gorie de cellules tres particuli~res chez les Gorgoncs (Ccelent6r6s octocorMIiaires). Vie Milieu, 19, (l, A), 5q 68. BOULIGAND, Y, 1969. Sur Fexistence de pseudomorphoses cholestdriques chez divers organismes vivants, a. Physique (Coll. C43, suppl, au 11 12, 30, 90 103. BOULIGAND, Y. and PUISEUX~DAo, S. I966. Sur I'arrangement des filaments d'A.D.N, dans les chromosomes de P6ridiniens et de certaines Bactdries. Sivth Intern. Congress Electron Mier., K)'oto, 2, 351 352. BOULIGAND, Y., S()YER, M.-O. and PU~SEUX-DAo, S. 1969 La structure fibrillaire et l'orientation des chromosomes chez les Dinoflagellds. Chromosoma, 24, 251 287. BOUNHH)I, J. J. and RATEL, R. 1964. Modifications exp6rimentales de l'aspect et de la structure de l'exuvie (retie nymphale/chez ,gombyv mori. C. r. kebd. Sdanc. Acad. Sci., Paris, 259, 914- 916. BOYDE, A. and MOBDH,, M. M. 1969. Scanning electron Microscopy of Lamellar Bone. Z. ZeHforsch. mikrosk. Anat., 93, 213-231. CHAFE, S. C. 1970. The fine structure of the Collenchyma Cell Wall. Plattta, 90, 12-2I. CHAPMAN, D. M, 1968. Structure histochemistry and formation of the podocyst and cuticle of .4ttrelia aarita. J. mar. biol. Ass. U.K,, 48, 187-208. CHISTYAKOV, 1. G. 1967. Liquid crystals. Soviet Phys. Uspeski, 9, 551-573. DELA~N, E. and RIOU, G. 1968. Uhrastructure des alt6rations du DNA du kin6,toplaste de Trypanosoma crllzi, trait6 par le bromure d'~thidium. C. r. hebd. Sdanc. Acad. Sc;., Paris', 268, 1327 1330. DENNELL, R. 1960. Integument and Exoskeleton. In Tbe Physiology el Crastacea, ed. by T. M. Waterman. Academic Press, 1, 449-472. DICKSON, M. R. and MERCER, E. H. 1967. Fine structural changes accompanying desiccation in philodhla roseola (Rotifera), J. Microscopic, 6, 331 348. D()BI.ER, J. and BERTLES, J. lz. 1968. The physical state of hemoglobin in sickle-cell anemia erythrocytes in rive. J. exp. Med., 127, 711-716. DODGt, J. 1964. Chromosome structure in the Dinophyceae, II. Cytochemical studies. Arch. Mil,-robiol., 48, 66 80. DRACH, P. 1939. Mue et cycle d'intermue chez les Crustac6s d6capodes. Ann. Inst. Ocdan., 19, Fasc. 3, 103-392. DRAC*q, P. 1953. Structure des lamel!es cuticulaires chcz les Crustac6s. C.r. hebd. Sc;anc. Aead. Sci., Paris, 237, 1772-I774. FRANK, R., FRANK, P., KLHN, M. and FONTMNE, R. 1959. Microscopie 61ectronique de l'os humain. Archs. Atiat. mierosc. Morph. exp. 44, 191-206. FREY-W1JSSLING, A. 1953. Submicroscopic morphology o f Protoplasm. Elsevier Publ. Co., Amsterdam, 1,303. FRIED~L, M. G. 1922. Les dtats mOsomorphes de la mati&e. Ann. Phys., Paris, 9, 273-474.

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