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American Mineralogist; November 2006; v. 91; no. 11-12; p. 1937-1940; DOI: 10.2138/am.2006.2391
© 2006 Mineralogical Society of America
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Letter

Crystallographic alignments in a coccolith (Pleurochrysis carterae) revealed by electron back-scattered diffraction (EBSD)

Kazuko Saruwatari1,*, Noriaki Ozaki1, Hiromichi Nagasawa2 and Toshihiro Kogure1

1 Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
2 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan

Correspondence: * E-mail: kazuko{at}eps.s.u-tokyo.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Sample and methods
 Results and discussion
 Acknowledgments
 References cited
 
Crystal orientations of the sub-micrometer-sized calcite units in oval-shaped coccoliths of a coccolithophore, Pleurochrysis carterae, have been investigated using electron back-scattered diffraction (EBSD). Although the contrast of the acquired EBSD patterns was weak due to the small crystal unit size, the orientations could be uniquely determined from the patterns. The crystal orientations for V- and R-units are close to those reported in a previous work using electron diffraction in a TEM. However, more accurate crystal orientations corresponding to the coccolith morphology were obtained by using EBSD in a SEM. In V-units, the c-axis is declined about 35° from the normal of the coccolith plane and one of the ai-axes is roughly parallel to the coccolith plane. The c-axis in R-units is slightly oblique to the radial direction along the coccolith plane and one of the ai-axes is near vertical to the coccolith plane. The projections of the c-axis of V- and R-units on the coccolith plane deviate considerably from the normal of the coccolith circumference, giving a crystallographic chiral property. The atomic arrangements of calcite contacted with the organic base plate are discussed for both units based on the crystallographic orientations derived from EBSD measurements.

Key Words: Biomineralization • coccolith • EBSD • SEM • calcite


   INTRODUCTION
Top
 Abstract
 Introduction
Sample and methods
 Results and discussion
 Acknowledgments
 References cited
 
Coccoliths are an environmentally, biologically, and geologically important biomineral produced as calcified scales of marine unicellular algae (Coccolithophores). In general, a coccolith consists of several tens of calcite crystals that are interlocked together to form a ring or disk with a small size of 1–10 µm across and with elaborate species-specific morphology (e.g., Young and Henriksen 2003). A key approach to understand the biomineralization mechanism of coccoliths is the integrated comprehension of the crystallography and morphology of whole coccolith units. To determine the crystallographic orientations in coccoliths, polarizing light microscopy has primarily been used (e.g., Young et al. 1992) and electron diffraction (ED) in a transmission electron microscopy (TEM) has been conventionally applied for more precise studies (e.g., Mann and Sparks 1988; Didymus et al. 1994; Marsh 1999). However, rigorous determination of the crystallographic orientation by ED generally requires a pair of diffraction patterns for one crystal and the comprehension of the morphology by TEM is often difficult because a TEM image is just a projection of the crystal formed by transmitted electrons. Recently, computer-tomography (CT) in TEM is under development to overcome this disadvantage (Kübel et al. 2005), but still not common. Atomic force microscopy (AFM) has also been applied to investigate the relationship between the morphology and the crystallographic orientation in coccoliths (Young and Henriksen 2003; Henriksen et al. 2003, 2004). However, the acquisition of high-resolution periodic images is generally limited to atomically flat surfaces. In contrast, scanning electron microscopy (SEM) has been effective for understanding the morphology of the specimens. Development of the field-emission (FE) gun has improved the resolution of SEM considerably. Furthermore, crystallographic orientations can be determined in a SEM by the recent development of the electron back-scattered diffraction (EBSD) technique (Venables and Harland 1973). Consequently, it has become possible to determine the relationship between the crystallographic orientations and morphologies by combining FE-SEM and EBSD (e.g., Kameda et al. 2005).

In the present study, crystallographic orientations of V- and R-units in coccoliths of a coccolithophore, Pleurochrysis carterae, were successfully determined by using FE-SEM and EBSD. This is the first report to use EBSD to determine the crystal orientations in sub-micrometer-sized biominerals with elaborate morphologies [a part of this study was reported in the conference abstract (Ozaki and Kogure 2003)]. Based on the results obtained, the atomic geometry of calcite at the organic-inorganic interfaces in coccoliths is discussed.


   SAMPLE AND METHODS
Top
 Abstract
 Introduction
 Sample and methods
Results and discussion
 Acknowledgments
 References cited
 
The coccolith scales were isolated from mono-species algal cultures and cleaned by the removal of organic matter according to the method of Marsh et al. (1992). The processed coccoliths were suspended in deionized water and the suspension was dispersed on silicon wafers. Before the dispersion, the silicon wafers were covered with a thin Pt-Pd film to acquire the averaged intensity of the back-scattered electrons, which was subtracted from the EBSD patterns of the specimen to enhance the Kikuchi bands. The dispersed coccoliths on the wafer were coated with a carbon film to prevent electrical charging in the SEM.

The SEM and EBSD studies were carried out using a Hitachi S-4500 SEM with a cold FE gun and a ThermoNoran PhaseID EBSD system. The accelerating voltage was 20 kV and the typical specimen current was 1 to 2 nA to acquire EBSD patterns. The acquisition time was normally about ten seconds. Analyses of the acquired EBSD patterns were performed using a program developed by Kogure (2003). SEM micrographs of the surface relief were obtained by lowering the accelerating voltage to 2 kV.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Sample and methods
 Results and discussion
Acknowledgments
 References cited
 
P. carterae is a cricoliths-type coccolith, which is defined as an elliptical heterococcolith with elements arranged in a simple ring on a base plate scale (Heimdal 1993). This coccolith consists of two kinds of interlocked units on an oval organic base plate (Marsh 1994, 1999; Okazaki et al. 1998). The two units are referred to as V- and R-units, which represent the crystal units with sub-vertical and sub-radial orientations of the c-axis, respectively (Young et al. 1992, 1997; Marsh 1999). Two template models have been suggested to accommodate the alternating crystallographic V- and R-units. One is a plicated template model proposed by Young et al. (1992), the other is a folded ribbon model proposed by Marsh (1999). Figures 1a and 1bGo correspondingly show SEM images of a distal (side away from the cell) and a proximal (side facing the cell) view of coccoliths that are inclined by 70° from the horizontal and nearly faced to the EBSD detector. In the distal view (Fig. 1aGo) distal shields (d) and outer tubes (o) that consist of V-units are clearly observed with inner tubes (i) of R-units [see Young et al. (1997) about these terminologies for coccoliths]. Conversely in the proximal view (Fig. 1bGo), the proximal shield (p), composed of R-units, is exposed on the top of the coccolith. Since the SEM images were taken at a low voltage (2 kV), the organic base plate (b) is observed without penetration.


Figure 1
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  		FIGURE 1. SEM images of coccoliths viewed from (a) the distal and (b) the proximal sides with inclination of 70° from the horizontal. Insets in the top-right of both figures are schematic cross sections adapted from Marsh (1999). The elements colored with light blue and light pink indicate single V- and R-units, respectively. The distal shields (d), outer tube elements (o), organic base plate (b), proximal shields (p), and inner tube elements (i) are indicated in the figures. The arrow in a indicates the point from which the EBSD pattern in Figure 2Go was acquired.

 
For EBSD measurements on non-polished specimens, the analyzing points must be generally on the top of the object because the EBSD detector acquires back-scattered electrons within a wide angle (~70°) from the analyzing point. Hence, the distal and proximal views are suitable for analyzing the V- and R-units, respectively. The acquired EBSD pattern from one of the distal shield elements (the arrow in Fig. 1aGo) is shown in Figure 2aGo. The contrast of the EBSD pattern is not intense if compared with that acquired from large calcite crystals (Kogure 2003). This is owing to the fact that most electrons with high energy (20 KeV) penetrate the small calcite crystals of coccoliths without scattering and reach the Pt-Pd coated silicon substrate. However, several Kikuchi bands are clearly visible in Figure 2aGo (three bands are enough to calculate the crystal orientation) and the corresponding Kikuchi pattern could be unambiguously calculated (Fig. 2bGo). Figure 2cGo is the stereographic projection of the crystallographic axes determined by the EBSD analysis. The origin in the figure corresponds to the substrate normal. The SEM image in the Figure 1aGo implies that the coccolith is directly placed on the silicon substrate so that the coccolith plane is nearly parallel to the substrate. Thus, the origin in the projected figure is the normal to the coccolith plane as well as the substrate. This is one of the advantages in the present SEM-EBSD analysis compared with TEM analyses in which it is difficult to recognize the precise attitude of coccoliths from the projected images.


Figure 2
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  		FIGURE 2. (a) EBSD pattern acquired from the arrowed point in Figure 1aGo. (b) Calculated Kikuchi pattern corresponding to the EBSD pattern in a. (c) Stereographic projections of [100], [Formula0], and [001] directions, showing the crystal orientation determined by EBSD analysis. The origin in the stereo net corresponds to the normal of the substrate. (d) Top view of the coccolith in Figure 1aGo. The arrows are projections of [100] (orange) and [001] (red) directions superimposed on the analyzed V-units. The numbers are angles inclined upward from the horizontal surface.

 
Because calcite is in the trigonal system, a unique c-axis and three equivalent ai-axes (i = 1, 2, 3) exist in the structure. In the following discussion, we define the a1-axis as one of the ai-axes whose direction is the most nearly parallel (V-unit) or vertical (R-unit) to the coccolith plane and express its direction as [100]. To easily understand the relationship between the crystallographic and morphological orientations in a coccolith, the projections of [001] and [100] directions (Fig. 2cGo) are superimposed on the coccolith image viewed from the top of the substrate (Fig. 2dGo). The black and white numbers in Figure 2dGo correspondingly represent the angle of the [001] and [100] directions from the substrate plane.

Figures 3a and 3bGo show the crystallographic orientations of several V- and R-units, respectively. Note that the organic base plate was peeled away from the coccolith in Figure 3bGo. The [100] directions of V-units (Fig. 3aGo) are nearly parallel to the coccolith plane with 8 ± 3° upward inclinations. The projection of the [100] directions onto the coccolith plane make angles of about 30° (anti-clockwise rotation) from the local tangent to the coccolith circumference. All [001] directions of V-units incline upward with 55 ± 3° from the coccolith plane. Their projections onto the coccolith plane were generally oriented to the direction about 50° (clockwise rotation) from the local tangent to the coccolith circumference. These results are generally consistent with those reported by Marsh (1999), but two findings are different. First, the projection of the [001] direction onto the coccolith plane has the opposite orientation with respect to the local tangent of the coccolith rim. Secondly, the normal direction of the V-units to the coccolith plane is approximately <Formula1> in our results, whereas it was <Formula1>in her result.


Figure 3
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  		FIGURE 3. SEM images of coccoliths observed perpendicularly from the (a) distal and (b) proximal side. The arrows are the same ones in Figure 2dGo, showing the projections of [100] and [001] directions determined by the EBSD analyses.

 
In the case of R-units (Fig. 3bGo), the [001] directions are nearly parallel to the coccolith plane with 10 ± 3° upward from the plane and their projections on the coccolith plane are oriented to the direction of 80 ± 10° (clockwise) from the local tangent to the coccolith circumference. On the other hand, the [100] directions are oriented closely (80 ± 4°) to the normal of the coccolith plane. Comparing with the results by Marsh (1999), the [001] directions are neither fully parallel nor radial to the coccolith plane. The normal directions of R-units to the coccolith plane are oblique to the <Formula00> direction with 10 ± 3°.

These results indicate three-dimensional regulation of crystallographic orientations for both V- and R-units in P. carterae, as also observed in E. huxleyi (Mann and Sparks 1988; Davis et al. 1995) and other species (Young et al. 1999). Furthermore, it has been clearly revealed that the alignments of V- and R-units have crystallographic chiral aspects, although morphological chirality is not obvious in P. carterae. Chiral morphologies are distinct in E. huxleyi and Coccolithus (Young et al. 1999; Young and Henriksen 2003). Didymus et al. (1994) also documented consistent crystallographic chirality in E. huxleyi. The systematic crystallographic orientations in the protococcolith suggest that the nucleation on the organic base plate is responsible for these orientations with the chiral aspects (Young and Henriksen 2003). In the case of P. carterae, the normal directions of V- and R-units to the coccolith plane are approximately <Formula1> and 10 ± 3° oblique to [Formula00], respectively, so that the contacting surfaces to the organic base plate are calculated to be almost {1Formula 24} and {2Formula1}, correspondingly. The {1Formula 24} plane is close to {0Formula4} with the difference of about 10°, while the {2Formula1} plane is close to{2<Formula0} with the difference of about 8°. The atomic arrangements on the {0Formula4} and {2Formula0} planes have a similar aspect in that theCa and C atoms are separately aligned with the distance of 4.05 Å in each row separately and alternatively lined in another row with the distance of 6.42 Å (Fig. 4Go). Remarkably, the alternating rows of Ca and C atoms with the 6.42 Å periodicity in both units are nearly parallel to the coccolith rim. The atomic arrangement in the {2Formula0} plane viewed along the [100] direction is also suggested as the interface to the organic substrate in E. huxleyi by Mann and Sparks (1988). Although the organic base plate has not been fully clarified, it was suggested that it was made of concentric and radial celluloses immersed into an amorphous matrix (Brown et al. 1969, 1970). The complex of the cellulose and the amorphous materials might be associated with the alternating atomic alignments with the 6.42 Å periodicity.


Figure 4
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  		FIGURE 4. Atomic arrangements of Ca (filled circles) and C (smaller open circles) on the calcite {0Formula4} and {2Formula0} planes that are close to the bottom planes of the V- and R-units, respectively, and so are possibly in contact with the organic base plate. The {2Formula0} plane is viewed along the [100] direction. The directions of the coccolith rim are also shown as broken lines.

 


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Sample and methods
 Results and discussion
 Acknowledgments
References cited
 
This work was supported by a Grant-in-Aid for scientific research (No. 17GS0311) from the Ministry Education, Culture, Sports, Science and Technology of Japan. The high-resolution SEM and EBSD analyses were carried out in the Electron Microbeam Analysis Facility of Department of Earth and Planetary Science, the University of Tokyo.


   Footnotes
 
MANUSCRIPT HANDLED BY BRYAN CHAKOUMAKOS

MANUSCRIPT RECEIVED July 19, 2006; MANUSCRIPT ACCEPTED August 21, 2006


   REFERENCES CITED
Top
 Abstract
 Introduction
 Sample and methods
 Results and discussion
 Acknowledgments
 References cited
 

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