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Cryo-electron microscopy reveals macromolecular organization within biological liquid cyrstals seen in the polarizing microscope
C ryo-electron microscopy reveals macromolecular organization within biological liquid crystals seen in the polarizing microscope F. P. Booy* and A. G. Fowler European Molecular Biology Laboratory, Postfach 10.2209, 6900 Heidelberg, FRG (Received 28 April 1985; revised 21 June 1985) /
A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf l and fd as examples. The technique reveals liquidcrystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packino of the virus particle is revealed to a spatial resolution of better than 30 A, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 A). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy. Keywords: Cryo-electronmicroscopy;liquid crystals; filamentousbacterial viruses; polarizing microscopy;X-ray fibrediffraction
Introduction Since liquid crystals were first formally described in 1888 ~ it has become well established that many biological materials exist in, or can be induced to form such, states of cohesion. The nomenclature of liquid crystal phases is complex but fully described in Demus and Richter 2 and De Gennes a. Nematic, cholesteric (a subclass of the nematic phase, No) and smectic textures will be described here. Other terms used include tactoids or, more formally, cybotactic groups which are small domains with a local smectic organization (see De Gennes 3 p. 329). Streamers, sometimes referred to as 'bloomers', are recognized in the cholesteric phase as regions of uniform iridescence in the polarizing microscope. Defects seen in smectic textures are more formally referred to as disclinations, that is disturbances in the texture which leave an enclosed volume of perfect fluid (in this case buffer). Dendrites or batonettes are textures that are extended in one direction. Many lyotropic (i.e. water dispersible) biomacromolecules and viruses provide splendid examples of liquid crystal behaviour4-7 ; as such they have been extensively investigated by polarizing optical microscopy to a resolution limited only by the thickness and uniformity of the specimen and ultimately by the instrument itself. From sols of some such materials it is possible to make fibres which are ideally suited for study by X-ray fibre diffraction 8 ,0 and structural data can be obtained to the 2-10 A level depending on the orientational perfection. In many cases the region between these two techniques has never been adequately bridged. Even where electron microscopy of negatively stained dispersions of material * Present address and address for reprint requests: Biochemisch Laboratorium, Nijenborgh 16, 9747 AG Groningen,The Netherlands 0141 8130/85/060327-09503.00 ~" 1985 Butterworth& Co. [Publishers) Ltd
has been carried out the correlation with optical and Xray data is often rather less than perfect. We present here details of techniques that allow precise correlation between optical, X-ray and e.m. data for some fibrous, water-dispersible materials using the filamentous bacterial viruses fdt and Pfl as examples. It is shown that the examination of the optical characteristics of liquid crystal states can be extended to the determination of the molecular orientation by electron microscopy and that diffraction and imaging data lead to interesting and precise correlations with X-ray data.
Materials and methods Samples of the bacterial viruses were extracted and purified as described previously ~2 and were kindly provided by K. Zimmerman and the group of D. Marvin. Virus solutions were stored at concentrations of 510 mg ml- 1 in 50 mM Tris-HC1 with 5 mM sodium azide. Before use samples were dialysed overnight against 10 mM Tris at neutral pH and then concentrated by centrifugation. Prior to concentration samples were sometimes diluted to 1-2mgm1-1. The pellet of concentrated material was then softened overnight with buffer to give the required final concentration. The concentration of virus in sols and gels was determined spectrophotometrically at a wavelength of 270 nm, using absorption coefficients of 1.99 cm 2 mg- 1 and 3.84 cm 2 mg- ~ for Pfl and fd respectively 13 Optical characterization of t he material was performed using a Leitz Ortholux polarizing microscope (Pal I| BK) equipped with a graduated rotation stage. A Zeiss photo+ Some preliminary e,m. data have been presented beforefor fd~1.
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Cryo-electron microscopy of liquid crystals: F. P. Bogy and A. G. Fowler microscope with polarization (Nomarski) optics was also used. Electron microscopy was carried out with a Philips EM 400 fitted with an improved blade-type anti-contamination device and a liquid nitrogen cooling holder (Philips type PW 6591) used as described in Reference 14. Samples for X-ray diffraction were the fibres used in Reference 12 and other similar fibres. Samples for optical microscopy were the fibres used for X-ray diffraction and gels and sols (10 mg ml- ~< concentration used < 30 mg ml- 1) which were examined between the flattened walls of thin quartz-glass capillaries before and after treatment in a 7 Tesla magnetic field ~5 (Oxford Instruments, UK). Specimens for electron microscopy were prepared by transferring drops to carbon coated grids using wide-bore glass tubes to avoid as far as possible disturbing the developed liquid crystal textures. These grids were then either concentrated in sealed containers, with the humidity controlled by saturated salt solutions 16, at temperatures ranging from room temperature to 60°C, or used without further treatment. The prepared grids were frozen either directly in liquid nitrogen, or by projection into liquid ethane itself cooled in liquid nitrogen, transferred to the cooling holder under liquid nitrogen and examined frozen at ~ - 150°C. E.m. samples were also prepared from the specimens examined in the optical microscope as described above. Other samples were concentrated in the temperature and humidity controlled environment of the 7 Tesla magnet. Specimens were examined at 100 or 120 kV under low-dose conditions and micrographs were recorded on Ilford Ilfoset (FT 35) or Kodak 4463 emulsions with a total dose of 5-10 e A- 2.
application of a magnetic field to a sol in the state of Figure la aligns the growing batonettes which fuse laterally as can be seen in the optical micrograph Figure ld. The electron micrograph, Figure 3a, shows a uniform smectic texture extending over a large area and is comparable with the optical micrographs shown in Figures lc and ld. In Figure le the texture shown extends throughout the depth of the fibre. A close inspection of Figure 3a reveals that within each lamella bundles of filaments are angled to the layer normal. This is particularly emphasized by the lines of disclination (disclinations) which tend to begin and end at lamellar boundaries in this texture; this is also seen in Figure 2c (arrow). An optical transform (not shown) of a micrograph of part of Figure 3a gave clear 55, 32.6 and 27.5 A equatorial reflections and is consistent with the hexagonal packing of a cylinder 64 A in diameter. Electron diffraction confirms that the structure is preserved to high resolution. Figure 4 shows the electron diffraction pattern from a specimen of Pfl with sharp reflections extending to better than 4 A. The pattern, with strong off-meridional intensity at 5 A and equatorial intensity at 10 A, is characteristic of an or-helical structure with 5.4 units/turn. Although inelastic scattering obscures many of the lowangle reflections, it is nevertheless clear that the room temperature form has been maintained (cf. Plates 2a and 2f in Reference 12). Figure 5 shows a polarizing optical micrograph of a fibre of Pfl displaying clear bi-axial properties; this is to be compared with the angling noted in the electron micrograph Figure 3a. The X-ray fibre-diffraction patterns of this sample showed clear double orientation (not shown).
Results
Discu~ion
The development of condensed liquid crystal textures in the formation of highly crystalline fibres as seen in the polarizing optical microscope has been presented before for Pfl 15. With the filamentous virus fd the process follows a similar pattern. At a concentration of less than ~0.1 mg ml- ~ solutions are isotropic and thus no iridescence is seen in the optical microscope. Above this concentration these rod-like particles, with a mean interparticle separation smaller than the mean length (the axial ratio is 150 for Pfl and 75 for fd), undergo a transition to a nematic state. This is the starting phase for the solutions used in this work. Higher concentrations resulted in the growth of smectic subphases. This is illustrated for Pfl in the optical micrograph Figure la, which shows cybotactic groups that are seen as randomly oriented inclusions in a bulk nematic phase. These have the appearance of Figure 2a in the electron microscope where in the case of fd the banding is alternately light and dark. The less dense advanced streamers that are seen as a uniform iridescence in the optical micrograph, Figure lb, envelop the original tactoids which act as nucleation centres for the development of dendrites or batonettes. This later stage of development is shown in the optical micrographs, Figures lc and d. The electron micrograph, Figure 2c, with fibres displaying a well defined 1 #m banding and containing in places a 55 A lateral periodicity is to be compared with the optical micrograph of Figure ld. The advanced streamers, in the smectic phase, with the 2.2/~m banding are growing at the expense of the nematic phase. The
The electron micrographs presented in Figures 2-4 demonstrate the power of cryo-electron microscopy to freeze-in and reveal the biological molecular associations within liquid crystal textures. This is in itself very interesting since previous attempts using conventional e.m. methods have not permitted direct correlation with the ultrastructural textures, for example, chromosomes from the dinoflagellates17. Although the various stages in textural development, for example, the initial nucleating tactoids, Figure 2a and the resulting dendrites, Figure 2c, can be seen by this direct method, it is easier to follow the dynamic effects of nucleation and rapid elongation of the dendrites by continuous observation in the optical microscope (Figure lc). Nucleation begins at cybotactic groups (Figure la, arrow) which were identified as inclusions with a higher numerical birefringence than the surrounding matrix. Streamers develop from these nucleation centres (Figure ld, arrow). Elongation occurs because a rod-like particle is able to diffuse easily in a direction parallel to its long axis. This phenomenon has parallels with the second step of the reptation process in the solid polymer melt ~8. A good example of a liquid crystal texture with clear disclinations is shown in Figure 3b. Here the liquid crystal texture shows a periodicity that is of the same order as the virion length ~3 and this strongly suggests a molecular origin to this mesophase with the virions packed approximately perpendicularly to the lamellae, thus forming a smectic texture (compare with Figure 62 in Reference 2).
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
la
Figure la (see legend overleaf)
lb
Figure lb-le (see legend overleaf)
1¢
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler Figure la (previous page) Sample of Pfl at a concentration of ~ 40 mg ml- 1 prepared by allowing a centrifuged pellet of material to resuspend overnight in an equal volume of supernatant. The specimen was observed with crossed polars and parallel (Krhler) illumination in a quartz glass capillary with flattened walls. The cross hairs indicate the direction of the polarizer (horizontal) and the analyser (vertical). Smectic phase cybotactic groups (arrow) are seen as inclusions that are randomly oriented in the bulk nematic (cholesteric) phase. The banding that is visible within the inclusions is ~ 2/~m. Scale bar = 20/~m Figure l b - l c (previous page) Sample of Pfl at a concentration of ~ 150mgml -~ prepared as in Figure la from the bottom of a centrifuged pellet of material taken up in a thin quartz glass capillary with flattened walls. (lb) Observed with crossed polars as in Figure la. Uniformly iridescent advanced streamers (bloomers) are seen in a bulk nematic phase. Close inspection of the original micrograph reveals that each streamer is a clearly defined smectic phase displaying a 2.2 gm periodicity. Scale bar = 20/~m. (lc) The same field of view as in ( I b) but viewed in schlieren contrast (with the analyser removed) enhancing the visibility of the 2.2 #m banding. Scale bar--- 20 pm
ld
Figure ld Optical micrograph of a sample of Pfl at a concentration of ~ 60 mg ml- 1 prepared from material at the bottom of a softening centrifuge pellet. The sample was taken up into a flattened quartz glass capillary and oriented in a 7 Tesla magnetic field for 10h at 10°C. The specimen was examined and photographed shortly after removal in a Zeiss photomicroscope equipped with Nomarsky interference contrast optics. Similar banding to that seen in Figure lc is visible in a dendritic smectic phase within the bulk nematic phase, The arrow points to the end of a nucleating tactoid. Scale b a r = 2 0 p m
le
Figure le Optical micrograph of a prepared X-ray fibre of iodinated Pfl ~~ ( # AF 221/3) examined between crossed polars (with a reduced substage condenser aperture). A 1.97 #m banding is visible. 1.96 ( + 7) #m was obtained using negatively stained dispersions. 23 Batonettes, with their long axis horizontal, run throughout the entire length of the fibre. The cross hairs indicate the direction of the polarizer (horizontal) and analyser (vertical). Scale bar = 20/~m
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2a Electron micrograph of a sample of fd concentrated by centrifugation in an airfuge, resuspended in an equal volume of supematant, transferred to a grid, quench-frozen in liquid nitrogen and examined frozen at ~ - 150°C in an electron microscope. An example ofa smectic, cybotactic group, quite commonlyfound in a nematic matrix is shown. The banding (light to dark band) is at 1 #m. Scale bar = 5 #m
The origin of the dark banding is not clear but may well be a result of head-to-head packing or a tilting of the virion. Asymmetry of the virion results from the A protein which is located at one end of the virion 19. The texture shown in Figure 2b is probably of nematic origin since the virion ends are not resolved (as in Figures 2c and 3a) but the lateral packing of the virions is seen over the entire field of view (see insert). However, Figure 3a is clearly a smectic texture and shows very long-range two-dimensional order. The disclinations (arrow) and thus the filaments are tilted with respect to the layer normals. Such a texture would give rise to bi-axial optical properties and is classed as the tilted smectic phase So. Indeed, the same feature is seen in optical studies of fibre specimens that give bi-axial X-ray diffraction patterns as is shown in Figure 5. Here the bundled virions maintain an unique orientation, as defined by the long axis of the indicatrix, for several tens of microns giving rise to a longrange structure reminiscent of the crimp observed in collagen fibres 2° in that a comparatively short molecule defines a texture with a much larger dimension. Figure 2c shows an association of dendrites (batonettes) in lateral register and illustrates the origin of disclinations at the lamellar boundaries, thus emphasizing the flexibility of the virions even in a block some pm 2 in size. The overlap with optical microscopy is particularly apparent at this
level where in Figure lda similar association of aligned textures is seen for Pfl. This association has been catalysed by the influence of a strong magnetic field resulting in the long-range order seen in two dimensions in the electron micrograph, Figure 3a, in the optical micrograph, Figure ld, and in three dimensions in the best fibres prepared for X-ray diffraction (optical micrograph Figure le). The size of the dendrites oriented by the magnetic field correlates well with the minimum mass (109 dalton) required for orientation in a 1 Tesla magnetic field 21. For both Pfl and fd it has been possible to produce highly crystalline X-ray fibres by means of magnetic alignment (e.g. Plate 2e, fibre # AF 221/3 in Reference 12). In the best such fibres of Pfl fine contiguous batonettes are revealed by careful optical microscopy. In Figure le the batonettes, which have developed from the lateral fusion of the advanced streamers (Figures lb and c) extend throughout the entire length of the fibre. However, the thickness of the fibre may result in an unreliable measurement of the periodicity along the batonette. For fd fibres it proved impossible to unambiguously measure the periodicity especially for fibres prepared at neutral pH, where considerably more orientational disorder is present. This is revealed as a twinning about the fibre axis in the X-ray diffraction pattern and is equivalent to the bi-
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2b Electron micrograph of a sample of fd prepared as for Figure 2a but before quench-freezing the prepared grid was partially dried at 60C at a controlled humidity in a 7 Tesla magnetic field for 48 h. The micrograph which is probably ofa nematic phase, shows a 55 A periodicity (insert shown boxed in white) over the entire field of view corresponding to the lateral packing offd viruses, scale bar = 10ttm. This is clearly revealed in the insert, scale bar--- 0.1 itm
axial disorder seen in Pfl. It is not possible to determine the molecular origin of such defects by studying the indicatrix of transverse sections alone, so it is rewarding that the electron micrographs, Fioures 2c and 3a, offer one possible explanation in terms of a molecular packing in the smectic phase. Indeed, until the electron micrograph, Figure 3a, was obtained it was generally believed that even the best X-ray fibres were composed of virions arranged randomly in the longitudinal direction, that is, in a nematic liquid crystal texture. Using our cryotechniques we have observed both nematic and smectic textures in sols of bacterial viruses. The twisted nematic subphase or cholesteric which was demonstrated by polarization microscopy was not clearly revealed by cryomicroscopy. This is due to the physical nature of cholesterics, namely that the helical pitch giving rise to characteristic iridescence is generally many times the diameter of the chiral solute molecule and thus could not be seen in the thin samples examined in this study. Cryosectioning of developed liquid crystal textures may well prove to be an alternative approach to the study of their structure. However, the systematic artefacts inherent in sectioning such as section compression and the possible requirement of a cryoprotectant to prevent freezing damage in larger samples 22 may complicate the interpretation of results. Wherever possible it is
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undoubtedly advantageous to freeze samples directly and to examine the aqueous textures with as few structural and textural modifications as possible. We consider that an extension of our techniques will be successful in examining the cholesteric phases in biological ultrastructures, such as the metaphase chromosome, chromosome folding and the cholesteric packing in the egg (chorion) of the silk moth, High voltage, low-dose cryomicroscopy will clearly be able to contribute to this field.
Conclusions The direct cryotechiques employed here have enabled us to visualize the molecular origin of the various liquid crystal textures that are required in the preparation of first class fibres for X-ray fibre diffraction. A means is now available to explore the reasons for some materials producing only poor fibres for X-ray diffraction and to follow the complex processes involved in the magnetic orientation of lyotropic specimens. Since the volumes required for electron microscopy are small when compared with X-ray diffraction it may be possible to provide structural data from small well developed textures for difficult or bi-axial specimens. The demonstration that high resolution selected area electron diffraction patterns can be obtained suggests that by imaging similar regions it
Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2e Electron micrograph of a sample offd prepared as for Figure2b. Clear batonette-like structures are seen which locally show a high degree of order with the 55 A filament separation resolved on the original negative within the well-defined 0.86/~m bands. The arrow points to the origin of a disclination in the texture. Scale bar = 1 #m
Figure 3a Electron micrograph of a sample offd prepared as for Figure 2b. A 0.83/zm banding is seen throughout a large region of the specimen which clearly has a smectic texture. Close inspection reveals the presence of disclinations beginning and ending at the lamellar boundaries (arrow). The disclinations are tilted with respect to the layer normals. Such a texture would give rise to bi-axial optical properties and would be classed as (So). Scale bar = 5 #m
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Cryo-electron microscopy o f liquid crystals: F. P. Boo 3, and A. G. F o w l e r
Figure 3b Electron micrograph of a sample of fd prepared as for Figure 2a. It is a good example of a smectlc hquld crystal texture showing a number of disclinations (arrow). Scale bar = 5 pm
may prove possible to obtain phased data directly from electron micrographs to supplement X-ray fibre diffraction data. These cryotechniques are directly applicable not only to in vitro systems but also to the many liquid crystalline textures that spontaneously develop in biological systems.
Acknowledgements We than Drs K. R. Leonard and P. Tucker for helpful discussion and criticism of the text. Dr R. Bryan helped in discussion.
References 1 2 3 4 5 6 7 8 9 10
Figure 4
Example of an electron diffraction pattern from a specimen of Pfl prepared as for the sample offd used in Figure 2a. The fibre axis is vertical and the pattern is of the room temperature form. The arrow indicates the 15th layer-line at a dspacing of 5.0 A
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11 12
Reinitzer, F. Monatsh. Chem. 1888, 9, 421 Demus, D. and Richter, L. in 'Textures of Liquid Crystals'. Weinheim, Verlag Chemie, 1978 De Gennes, P. G. in 'The Physics of Liquid Crystals', Clarendon Press, Oxford, 1975 Gregory J. and Holmes, K. C. J. Mol. Biol. 1965, 13, 796 Robinson, C. Molecular Crystals 1966, 1,467 Willison, J. H. M. J. UItrastruc. Res. 1976, 54, 176 Lapointe, J. and Marvin, D. A. J. Mol. Biol. 1973, 19, 269 Franklin, R. E. and Holmes, K. C. Acta. Crystallogr. 1958, I l, 213 Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. and Hamilton, L. D. J. Mol. Biol. 1960, 2, 19 Marvin, D. A., Wiseman, R. L. and Wachtel, E. J. J. Mol. Biol. 1974, 82, 121 Booy, F. P. Paper presented at the 10th International Congress on Electron Microscopy, Hamburg, 1982, p. 113 Nave, C., Brown, R. S., Fowler, A. G., Ladner, J. E., Marvin, D. A., Provencher, S. W., Tsugita, A., Armstrong, J. and Perham, R. N. J. Mol. Biol. 1981, 149, 675
Cryo-electron microscopy o f liquid crystals: F. P. Booy and A. G. Fowler
Figure 5 Prepared X-ray fibre of native Pfl viewed in the polarizing microscope. The fibre axis (arrow) is perpendicular to the lamellae in which the indicatrix is shown. The virion direction corresponds to the long axis of the indicatrix. The polarizer direction was horizontal and in order to enhance contrast the analyser was rotated 5 degrees clockwise from the vertical position. Scale bar = 20/~m
13 14 15
16 17
Wiseman, R. L., Berkowitz, S. A. and Day, L. A. J. Mol. Biol. 1976, 102, 549 Booy, F. P. and Van Bruggen, E. F. J. UItramicroscopy 1984, 13, 337 Fowler, A. G., Brown, R. S. and J~ickle, H. in 'Magnetic Field Effects on Bacterial Viruses and Polytene Chromosomes from Chironomus tentans', poster #TH-J-6 presented at the 7th International Biophysics Congress, Mexico City, 1981 Spenser, H. M. International Critical Tables 1926, 1, 67 Livolant, F. and Bouligand, Y. Chromosoma Berl. 1968, 80, 97
18 19 20 21 22 23
Cohen-Addad, J. P. Polymer 1983, 24, 1128 Grey, C., Brown, R. S. and Marvin, D. A. J. Mol. Biol. 1981, 146, 621 Gathercole, L. J. and Keller, A. in 'Colston Papers', (Eds E. D. T. Atkins and A. Keller), Butterworths, London, 1974, p. 153 Worcester, D. L. Proc. Natl. Acad. Sci. USA 1975, 175, 5475 Chang, J. J., McDowall, A. W., Lepault, J., Walter, C. A. and Dubochet, J. J. Microscopy 1983, 132, 109 Wiseman, R. L. and Day, L. A. J. MoI. Biol. 1977, 116, 607
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A method is described for examining water dispersible biopolymers in the frozen, hydrated state by electron microscopy using the filamentous bacterial viruses Pf l and fd as examples. The technique reveals liquidcrystalline textures that correlate well with polarizing microscopy of magnetically oriented specimens. At higher magnification the packino of the virus particle is revealed to a spatial resolution of better than 30 A, thus linking directly with data from X-ray diffraction and optical microscopy. Electron diffraction confirms that the structure is preserved to high resolution (4 A). The technique permits a detailed understanding of the processes involved in the orientation of these samples in a strong magnetic field and clarifies the long-range bi-axial properties of some fibres as seen by X-ray diffraction and optical microscopy. Keywords: Cryo-electronmicroscopy;liquid crystals; filamentousbacterial viruses; polarizing microscopy;X-ray fibrediffraction
Introduction Since liquid crystals were first formally described in 1888 ~ it has become well established that many biological materials exist in, or can be induced to form such, states of cohesion. The nomenclature of liquid crystal phases is complex but fully described in Demus and Richter 2 and De Gennes a. Nematic, cholesteric (a subclass of the nematic phase, No) and smectic textures will be described here. Other terms used include tactoids or, more formally, cybotactic groups which are small domains with a local smectic organization (see De Gennes 3 p. 329). Streamers, sometimes referred to as 'bloomers', are recognized in the cholesteric phase as regions of uniform iridescence in the polarizing microscope. Defects seen in smectic textures are more formally referred to as disclinations, that is disturbances in the texture which leave an enclosed volume of perfect fluid (in this case buffer). Dendrites or batonettes are textures that are extended in one direction. Many lyotropic (i.e. water dispersible) biomacromolecules and viruses provide splendid examples of liquid crystal behaviour4-7 ; as such they have been extensively investigated by polarizing optical microscopy to a resolution limited only by the thickness and uniformity of the specimen and ultimately by the instrument itself. From sols of some such materials it is possible to make fibres which are ideally suited for study by X-ray fibre diffraction 8 ,0 and structural data can be obtained to the 2-10 A level depending on the orientational perfection. In many cases the region between these two techniques has never been adequately bridged. Even where electron microscopy of negatively stained dispersions of material * Present address and address for reprint requests: Biochemisch Laboratorium, Nijenborgh 16, 9747 AG Groningen,The Netherlands 0141 8130/85/060327-09503.00 ~" 1985 Butterworth& Co. [Publishers) Ltd
has been carried out the correlation with optical and Xray data is often rather less than perfect. We present here details of techniques that allow precise correlation between optical, X-ray and e.m. data for some fibrous, water-dispersible materials using the filamentous bacterial viruses fdt and Pfl as examples. It is shown that the examination of the optical characteristics of liquid crystal states can be extended to the determination of the molecular orientation by electron microscopy and that diffraction and imaging data lead to interesting and precise correlations with X-ray data.
Materials and methods Samples of the bacterial viruses were extracted and purified as described previously ~2 and were kindly provided by K. Zimmerman and the group of D. Marvin. Virus solutions were stored at concentrations of 510 mg ml- 1 in 50 mM Tris-HC1 with 5 mM sodium azide. Before use samples were dialysed overnight against 10 mM Tris at neutral pH and then concentrated by centrifugation. Prior to concentration samples were sometimes diluted to 1-2mgm1-1. The pellet of concentrated material was then softened overnight with buffer to give the required final concentration. The concentration of virus in sols and gels was determined spectrophotometrically at a wavelength of 270 nm, using absorption coefficients of 1.99 cm 2 mg- 1 and 3.84 cm 2 mg- ~ for Pfl and fd respectively 13 Optical characterization of t he material was performed using a Leitz Ortholux polarizing microscope (Pal I| BK) equipped with a graduated rotation stage. A Zeiss photo+ Some preliminary e,m. data have been presented beforefor fd~1.
Int. J. Biol. Macromol., 1985, Vol 7, December
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Cryo-electron microscopy of liquid crystals: F. P. Bogy and A. G. Fowler microscope with polarization (Nomarski) optics was also used. Electron microscopy was carried out with a Philips EM 400 fitted with an improved blade-type anti-contamination device and a liquid nitrogen cooling holder (Philips type PW 6591) used as described in Reference 14. Samples for X-ray diffraction were the fibres used in Reference 12 and other similar fibres. Samples for optical microscopy were the fibres used for X-ray diffraction and gels and sols (10 mg ml- ~< concentration used < 30 mg ml- 1) which were examined between the flattened walls of thin quartz-glass capillaries before and after treatment in a 7 Tesla magnetic field ~5 (Oxford Instruments, UK). Specimens for electron microscopy were prepared by transferring drops to carbon coated grids using wide-bore glass tubes to avoid as far as possible disturbing the developed liquid crystal textures. These grids were then either concentrated in sealed containers, with the humidity controlled by saturated salt solutions 16, at temperatures ranging from room temperature to 60°C, or used without further treatment. The prepared grids were frozen either directly in liquid nitrogen, or by projection into liquid ethane itself cooled in liquid nitrogen, transferred to the cooling holder under liquid nitrogen and examined frozen at ~ - 150°C. E.m. samples were also prepared from the specimens examined in the optical microscope as described above. Other samples were concentrated in the temperature and humidity controlled environment of the 7 Tesla magnet. Specimens were examined at 100 or 120 kV under low-dose conditions and micrographs were recorded on Ilford Ilfoset (FT 35) or Kodak 4463 emulsions with a total dose of 5-10 e A- 2.
application of a magnetic field to a sol in the state of Figure la aligns the growing batonettes which fuse laterally as can be seen in the optical micrograph Figure ld. The electron micrograph, Figure 3a, shows a uniform smectic texture extending over a large area and is comparable with the optical micrographs shown in Figures lc and ld. In Figure le the texture shown extends throughout the depth of the fibre. A close inspection of Figure 3a reveals that within each lamella bundles of filaments are angled to the layer normal. This is particularly emphasized by the lines of disclination (disclinations) which tend to begin and end at lamellar boundaries in this texture; this is also seen in Figure 2c (arrow). An optical transform (not shown) of a micrograph of part of Figure 3a gave clear 55, 32.6 and 27.5 A equatorial reflections and is consistent with the hexagonal packing of a cylinder 64 A in diameter. Electron diffraction confirms that the structure is preserved to high resolution. Figure 4 shows the electron diffraction pattern from a specimen of Pfl with sharp reflections extending to better than 4 A. The pattern, with strong off-meridional intensity at 5 A and equatorial intensity at 10 A, is characteristic of an or-helical structure with 5.4 units/turn. Although inelastic scattering obscures many of the lowangle reflections, it is nevertheless clear that the room temperature form has been maintained (cf. Plates 2a and 2f in Reference 12). Figure 5 shows a polarizing optical micrograph of a fibre of Pfl displaying clear bi-axial properties; this is to be compared with the angling noted in the electron micrograph Figure 3a. The X-ray fibre-diffraction patterns of this sample showed clear double orientation (not shown).
Results
Discu~ion
The development of condensed liquid crystal textures in the formation of highly crystalline fibres as seen in the polarizing optical microscope has been presented before for Pfl 15. With the filamentous virus fd the process follows a similar pattern. At a concentration of less than ~0.1 mg ml- ~ solutions are isotropic and thus no iridescence is seen in the optical microscope. Above this concentration these rod-like particles, with a mean interparticle separation smaller than the mean length (the axial ratio is 150 for Pfl and 75 for fd), undergo a transition to a nematic state. This is the starting phase for the solutions used in this work. Higher concentrations resulted in the growth of smectic subphases. This is illustrated for Pfl in the optical micrograph Figure la, which shows cybotactic groups that are seen as randomly oriented inclusions in a bulk nematic phase. These have the appearance of Figure 2a in the electron microscope where in the case of fd the banding is alternately light and dark. The less dense advanced streamers that are seen as a uniform iridescence in the optical micrograph, Figure lb, envelop the original tactoids which act as nucleation centres for the development of dendrites or batonettes. This later stage of development is shown in the optical micrographs, Figures lc and d. The electron micrograph, Figure 2c, with fibres displaying a well defined 1 #m banding and containing in places a 55 A lateral periodicity is to be compared with the optical micrograph of Figure ld. The advanced streamers, in the smectic phase, with the 2.2/~m banding are growing at the expense of the nematic phase. The
The electron micrographs presented in Figures 2-4 demonstrate the power of cryo-electron microscopy to freeze-in and reveal the biological molecular associations within liquid crystal textures. This is in itself very interesting since previous attempts using conventional e.m. methods have not permitted direct correlation with the ultrastructural textures, for example, chromosomes from the dinoflagellates17. Although the various stages in textural development, for example, the initial nucleating tactoids, Figure 2a and the resulting dendrites, Figure 2c, can be seen by this direct method, it is easier to follow the dynamic effects of nucleation and rapid elongation of the dendrites by continuous observation in the optical microscope (Figure lc). Nucleation begins at cybotactic groups (Figure la, arrow) which were identified as inclusions with a higher numerical birefringence than the surrounding matrix. Streamers develop from these nucleation centres (Figure ld, arrow). Elongation occurs because a rod-like particle is able to diffuse easily in a direction parallel to its long axis. This phenomenon has parallels with the second step of the reptation process in the solid polymer melt ~8. A good example of a liquid crystal texture with clear disclinations is shown in Figure 3b. Here the liquid crystal texture shows a periodicity that is of the same order as the virion length ~3 and this strongly suggests a molecular origin to this mesophase with the virions packed approximately perpendicularly to the lamellae, thus forming a smectic texture (compare with Figure 62 in Reference 2).
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
la
Figure la (see legend overleaf)
lb
Figure lb-le (see legend overleaf)
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler Figure la (previous page) Sample of Pfl at a concentration of ~ 40 mg ml- 1 prepared by allowing a centrifuged pellet of material to resuspend overnight in an equal volume of supernatant. The specimen was observed with crossed polars and parallel (Krhler) illumination in a quartz glass capillary with flattened walls. The cross hairs indicate the direction of the polarizer (horizontal) and the analyser (vertical). Smectic phase cybotactic groups (arrow) are seen as inclusions that are randomly oriented in the bulk nematic (cholesteric) phase. The banding that is visible within the inclusions is ~ 2/~m. Scale bar = 20/~m Figure l b - l c (previous page) Sample of Pfl at a concentration of ~ 150mgml -~ prepared as in Figure la from the bottom of a centrifuged pellet of material taken up in a thin quartz glass capillary with flattened walls. (lb) Observed with crossed polars as in Figure la. Uniformly iridescent advanced streamers (bloomers) are seen in a bulk nematic phase. Close inspection of the original micrograph reveals that each streamer is a clearly defined smectic phase displaying a 2.2 gm periodicity. Scale bar = 20/~m. (lc) The same field of view as in ( I b) but viewed in schlieren contrast (with the analyser removed) enhancing the visibility of the 2.2 #m banding. Scale bar--- 20 pm
ld
Figure ld Optical micrograph of a sample of Pfl at a concentration of ~ 60 mg ml- 1 prepared from material at the bottom of a softening centrifuge pellet. The sample was taken up into a flattened quartz glass capillary and oriented in a 7 Tesla magnetic field for 10h at 10°C. The specimen was examined and photographed shortly after removal in a Zeiss photomicroscope equipped with Nomarsky interference contrast optics. Similar banding to that seen in Figure lc is visible in a dendritic smectic phase within the bulk nematic phase, The arrow points to the end of a nucleating tactoid. Scale b a r = 2 0 p m
le
Figure le Optical micrograph of a prepared X-ray fibre of iodinated Pfl ~~ ( # AF 221/3) examined between crossed polars (with a reduced substage condenser aperture). A 1.97 #m banding is visible. 1.96 ( + 7) #m was obtained using negatively stained dispersions. 23 Batonettes, with their long axis horizontal, run throughout the entire length of the fibre. The cross hairs indicate the direction of the polarizer (horizontal) and analyser (vertical). Scale bar = 20/~m
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2a Electron micrograph of a sample of fd concentrated by centrifugation in an airfuge, resuspended in an equal volume of supematant, transferred to a grid, quench-frozen in liquid nitrogen and examined frozen at ~ - 150°C in an electron microscope. An example ofa smectic, cybotactic group, quite commonlyfound in a nematic matrix is shown. The banding (light to dark band) is at 1 #m. Scale bar = 5 #m
The origin of the dark banding is not clear but may well be a result of head-to-head packing or a tilting of the virion. Asymmetry of the virion results from the A protein which is located at one end of the virion 19. The texture shown in Figure 2b is probably of nematic origin since the virion ends are not resolved (as in Figures 2c and 3a) but the lateral packing of the virions is seen over the entire field of view (see insert). However, Figure 3a is clearly a smectic texture and shows very long-range two-dimensional order. The disclinations (arrow) and thus the filaments are tilted with respect to the layer normals. Such a texture would give rise to bi-axial optical properties and is classed as the tilted smectic phase So. Indeed, the same feature is seen in optical studies of fibre specimens that give bi-axial X-ray diffraction patterns as is shown in Figure 5. Here the bundled virions maintain an unique orientation, as defined by the long axis of the indicatrix, for several tens of microns giving rise to a longrange structure reminiscent of the crimp observed in collagen fibres 2° in that a comparatively short molecule defines a texture with a much larger dimension. Figure 2c shows an association of dendrites (batonettes) in lateral register and illustrates the origin of disclinations at the lamellar boundaries, thus emphasizing the flexibility of the virions even in a block some pm 2 in size. The overlap with optical microscopy is particularly apparent at this
level where in Figure lda similar association of aligned textures is seen for Pfl. This association has been catalysed by the influence of a strong magnetic field resulting in the long-range order seen in two dimensions in the electron micrograph, Figure 3a, in the optical micrograph, Figure ld, and in three dimensions in the best fibres prepared for X-ray diffraction (optical micrograph Figure le). The size of the dendrites oriented by the magnetic field correlates well with the minimum mass (109 dalton) required for orientation in a 1 Tesla magnetic field 21. For both Pfl and fd it has been possible to produce highly crystalline X-ray fibres by means of magnetic alignment (e.g. Plate 2e, fibre # AF 221/3 in Reference 12). In the best such fibres of Pfl fine contiguous batonettes are revealed by careful optical microscopy. In Figure le the batonettes, which have developed from the lateral fusion of the advanced streamers (Figures lb and c) extend throughout the entire length of the fibre. However, the thickness of the fibre may result in an unreliable measurement of the periodicity along the batonette. For fd fibres it proved impossible to unambiguously measure the periodicity especially for fibres prepared at neutral pH, where considerably more orientational disorder is present. This is revealed as a twinning about the fibre axis in the X-ray diffraction pattern and is equivalent to the bi-
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Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2b Electron micrograph of a sample of fd prepared as for Figure 2a but before quench-freezing the prepared grid was partially dried at 60C at a controlled humidity in a 7 Tesla magnetic field for 48 h. The micrograph which is probably ofa nematic phase, shows a 55 A periodicity (insert shown boxed in white) over the entire field of view corresponding to the lateral packing offd viruses, scale bar = 10ttm. This is clearly revealed in the insert, scale bar--- 0.1 itm
axial disorder seen in Pfl. It is not possible to determine the molecular origin of such defects by studying the indicatrix of transverse sections alone, so it is rewarding that the electron micrographs, Fioures 2c and 3a, offer one possible explanation in terms of a molecular packing in the smectic phase. Indeed, until the electron micrograph, Figure 3a, was obtained it was generally believed that even the best X-ray fibres were composed of virions arranged randomly in the longitudinal direction, that is, in a nematic liquid crystal texture. Using our cryotechniques we have observed both nematic and smectic textures in sols of bacterial viruses. The twisted nematic subphase or cholesteric which was demonstrated by polarization microscopy was not clearly revealed by cryomicroscopy. This is due to the physical nature of cholesterics, namely that the helical pitch giving rise to characteristic iridescence is generally many times the diameter of the chiral solute molecule and thus could not be seen in the thin samples examined in this study. Cryosectioning of developed liquid crystal textures may well prove to be an alternative approach to the study of their structure. However, the systematic artefacts inherent in sectioning such as section compression and the possible requirement of a cryoprotectant to prevent freezing damage in larger samples 22 may complicate the interpretation of results. Wherever possible it is
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undoubtedly advantageous to freeze samples directly and to examine the aqueous textures with as few structural and textural modifications as possible. We consider that an extension of our techniques will be successful in examining the cholesteric phases in biological ultrastructures, such as the metaphase chromosome, chromosome folding and the cholesteric packing in the egg (chorion) of the silk moth, High voltage, low-dose cryomicroscopy will clearly be able to contribute to this field.
Conclusions The direct cryotechiques employed here have enabled us to visualize the molecular origin of the various liquid crystal textures that are required in the preparation of first class fibres for X-ray fibre diffraction. A means is now available to explore the reasons for some materials producing only poor fibres for X-ray diffraction and to follow the complex processes involved in the magnetic orientation of lyotropic specimens. Since the volumes required for electron microscopy are small when compared with X-ray diffraction it may be possible to provide structural data from small well developed textures for difficult or bi-axial specimens. The demonstration that high resolution selected area electron diffraction patterns can be obtained suggests that by imaging similar regions it
Cryo-electron microscopy of liquid crystals: F. P. Booy and A. G. Fowler
Figure 2e Electron micrograph of a sample offd prepared as for Figure2b. Clear batonette-like structures are seen which locally show a high degree of order with the 55 A filament separation resolved on the original negative within the well-defined 0.86/~m bands. The arrow points to the origin of a disclination in the texture. Scale bar = 1 #m
Figure 3a Electron micrograph of a sample offd prepared as for Figure 2b. A 0.83/zm banding is seen throughout a large region of the specimen which clearly has a smectic texture. Close inspection reveals the presence of disclinations beginning and ending at the lamellar boundaries (arrow). The disclinations are tilted with respect to the layer normals. Such a texture would give rise to bi-axial optical properties and would be classed as (So). Scale bar = 5 #m
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Cryo-electron microscopy o f liquid crystals: F. P. Boo 3, and A. G. F o w l e r
Figure 3b Electron micrograph of a sample of fd prepared as for Figure 2a. It is a good example of a smectlc hquld crystal texture showing a number of disclinations (arrow). Scale bar = 5 pm
may prove possible to obtain phased data directly from electron micrographs to supplement X-ray fibre diffraction data. These cryotechniques are directly applicable not only to in vitro systems but also to the many liquid crystalline textures that spontaneously develop in biological systems.
Acknowledgements We than Drs K. R. Leonard and P. Tucker for helpful discussion and criticism of the text. Dr R. Bryan helped in discussion.
References 1 2 3 4 5 6 7 8 9 10
Figure 4
Example of an electron diffraction pattern from a specimen of Pfl prepared as for the sample offd used in Figure 2a. The fibre axis is vertical and the pattern is of the room temperature form. The arrow indicates the 15th layer-line at a dspacing of 5.0 A
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Reinitzer, F. Monatsh. Chem. 1888, 9, 421 Demus, D. and Richter, L. in 'Textures of Liquid Crystals'. Weinheim, Verlag Chemie, 1978 De Gennes, P. G. in 'The Physics of Liquid Crystals', Clarendon Press, Oxford, 1975 Gregory J. and Holmes, K. C. J. Mol. Biol. 1965, 13, 796 Robinson, C. Molecular Crystals 1966, 1,467 Willison, J. H. M. J. UItrastruc. Res. 1976, 54, 176 Lapointe, J. and Marvin, D. A. J. Mol. Biol. 1973, 19, 269 Franklin, R. E. and Holmes, K. C. Acta. Crystallogr. 1958, I l, 213 Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. and Hamilton, L. D. J. Mol. Biol. 1960, 2, 19 Marvin, D. A., Wiseman, R. L. and Wachtel, E. J. J. Mol. Biol. 1974, 82, 121 Booy, F. P. Paper presented at the 10th International Congress on Electron Microscopy, Hamburg, 1982, p. 113 Nave, C., Brown, R. S., Fowler, A. G., Ladner, J. E., Marvin, D. A., Provencher, S. W., Tsugita, A., Armstrong, J. and Perham, R. N. J. Mol. Biol. 1981, 149, 675
Cryo-electron microscopy o f liquid crystals: F. P. Booy and A. G. Fowler
Figure 5 Prepared X-ray fibre of native Pfl viewed in the polarizing microscope. The fibre axis (arrow) is perpendicular to the lamellae in which the indicatrix is shown. The virion direction corresponds to the long axis of the indicatrix. The polarizer direction was horizontal and in order to enhance contrast the analyser was rotated 5 degrees clockwise from the vertical position. Scale bar = 20/~m
13 14 15
16 17
Wiseman, R. L., Berkowitz, S. A. and Day, L. A. J. Mol. Biol. 1976, 102, 549 Booy, F. P. and Van Bruggen, E. F. J. UItramicroscopy 1984, 13, 337 Fowler, A. G., Brown, R. S. and J~ickle, H. in 'Magnetic Field Effects on Bacterial Viruses and Polytene Chromosomes from Chironomus tentans', poster #TH-J-6 presented at the 7th International Biophysics Congress, Mexico City, 1981 Spenser, H. M. International Critical Tables 1926, 1, 67 Livolant, F. and Bouligand, Y. Chromosoma Berl. 1968, 80, 97
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Cohen-Addad, J. P. Polymer 1983, 24, 1128 Grey, C., Brown, R. S. and Marvin, D. A. J. Mol. Biol. 1981, 146, 621 Gathercole, L. J. and Keller, A. in 'Colston Papers', (Eds E. D. T. Atkins and A. Keller), Butterworths, London, 1974, p. 153 Worcester, D. L. Proc. Natl. Acad. Sci. USA 1975, 175, 5475 Chang, J. J., McDowall, A. W., Lepault, J., Walter, C. A. and Dubochet, J. J. Microscopy 1983, 132, 109 Wiseman, R. L. and Day, L. A. J. MoI. Biol. 1977, 116, 607
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