SCANNING VOL. 25, 12–18 (2003) Received: December 7, 1999© FAMS, Inc. Accepted with revision: December 17, 2002

Microstructure of Monoplacophora (Mollusca) Shell Examined by Low-Voltage Field Emission Scanning Electron and Atomic Force Microscopy

RENATO CRUZ, GILBERTO WEISSMÜLLER, MARCOS FARINA*

Instituto de Biofísica Carlos Chagas Filho; *Departamento de Anatomia, Instituto de Ciências Biomédicas, CCS, UniversidadeFederal do Rio de Janeiro, Rio de Janeiro, Brazil

Summary: The shell of Micropilina arntzi (Mollusca:Monoplacophora), a primitive molluscan class, was ex-amined by using field emission scanning electron mi-croscopy (FESEM) at low voltage and atomic force mi-croscopy (AFM). The use of these two techniques allowedthe observation of fine details of Micropilina arntzi shelland contributed to bring new features concerning the studyof molluscan shell microtexture. Imaging with low-voltageFESEM provided well-defined edge contours of shell struc-tures, while analyzing the sample with AFM gave infor-mation about the step height of stacked internal structuresas well as the dimension of the particles present in their sur-face at a nanometric level. The shell microstructure ofMonoplacophora species presents different patterns andmay be a taxonomic implication in the systematic studiesof the group.

Key words: field emission scanning electron microscopy,low voltage, atomic force microscopy, microstructure,mollusca

PACS: 87.64.Dz, 07.79.-v, 61.16.Fk

Introduction

The phylum Mollusca is the most abundant group amongthe invertebrates, with approximately 128,000 extantspecies. The majority of them (Conchifera) produce shellscomposed by polymorphs of calcium carbonates (CaCO3)intercalated by proteins and glycoproteins (Brusca and Br-usca 1990, Krampitz and Graser 1988). Calcite and arag-onite are the most common calcium carbonate phases

formed by mollusks (Lowenstam and Weiner 1989). Theshell may contain one phase or the other, or both, depend-ing on the species (Carter and Clarke 1985). However,some paleontological and phylogenetic evidences suggestthat the primitive molluscan shell was wholly aragonitic(Carter and Clarke 1985, Lowenstam and Weiner 1989).The shell is considered one of the most significant char-acters of the phylum Mollusca (Schaefer and Haszprunar1997). The early characterizations of shell microstructurein the mollusks were based on light microscopy (Bøggild1930). After the decade of 1960, electron microscopy be-came a new important tool in the determination of shell mi-crostructure (Kobayashi 1964, Taylor et al. 1969, Watan-abe 1988); more recently the atomic force microscope wasemployed for analysis at a nanoscale level (Giles et al.1995, Manne et al. 1994, Sikes et al. 1998).

The class Monoplacophora has been considered a keygroup for molluscan evolution (Haszprunar 1988, Ivanov1996, Runnegar 1996). In this class, a considerable num-ber of primitive characters was retained (Haszprunar 1988).The Monoplacophora were first known from fossil recordsfrom the early Cambrian to the Cenozoic period (Runnegarand Pojeta 1974), and until recently it had been assumed thatall species were extinct. However, in 1952, live monopla-cophoran specimens were discovered at a depth of approx-imately 4,000 meters in the eastern Pacific (Lemche 1957).

Due to the reduced numbers of specimens available,few studies have been published about the microstructureof fossil and extant Monoplacophora shells (Erben et al.1968, Taviani et al. 1990, Warén and Gofas 1996). In thepresent work, we examined the microstructure, texturepattern, and mineral composition of the shell of Micropilinaarntzi (Monoplacophora; Mollusca), a living specimen,by using field emission scanning electron microscopy(FESEM) operating at low voltage, electron spectroscopicdiffraction, and atomic force microscopy (AFM).

Materials and Methods

The specimens of Micropilina arntzi (Monoplacophora:Mollusca) were collected on February 17, 1991, by the R/V“Polarstern” in the Lazarev Sea (70°18.09’S × 03°15.08’Wto 70°19.02’S × 03°16.08’W),Antarctica, between 191 and

This work was supported by Brazilian grants from PRONEX, CAPESand Faperj.

Address for reprints:

Marcos Farina, Ph.D.Departamento de Anatomia,Instituto de Ciências Biomédicas, CCS,Universidade Federal do Rio de Janeiro,21941-590, Rio de Janeiro, RJ, Brasil.e-mail: mfarina@anato.ufrj.br

204 meters of depth. They were immediately fixed inbuffered formaldehyde at 4% for 24 h and stored in 70%ethanol.

Polarization Light Microscopy

The shells were rinsed in distilled water and dehydratedin ethanol. The shell was mounted in glass slides with syn-thetic balsam (Entellan; Merck, Darmstadt, Germany) andobserved in a Zeiss Axiophot microscope (Carl Zeiss,Oberkochen, Germany) between crossed polarizers.

Field Emission Scanning Electron Microscopy

Samples of shell were extensively washed in distilledwater, air dried, and mounted on appropriate specimensupports (Silicon chips, Pelco International, Redding,Calif., USA). The chips were placed on aluminum stubsand coated (3 nm) with chromium in a high-resolution ionbeam coater (Gatan, model 681, Oxford, U.K.). The spec-imens were examined in a Jeol 6340 FESEM (JEOL, Ak-ishima, Japan), operating at accelerating voltages rangingfrom 2 to 5 kV and imaged by using secondary electrons.

Atomic Force Microscopy

The AFM used in this work was built at the Instituto deBiofísica Carlos Chagas Filho, Universidade Federal doRio de Janeiro, as part of a collaboration with the Ludwig-Maximilian Universität, Lehrstuhl für Angewandte Physik,in Munich, Germany. Coverslips were glued to magneticstainless steel punches and mounted in a fluid cell. Sam-ples were scanned in contact error mode in air, at room tem-perature, and using a standard silicon nitride tip (Digital In-struments Inc., [now Veeco Metrology Group, SantaBarbara, Calif., USA]) with a 4 µm2 pyramidal basemounted on a V-shaped cantilever of spring constant 0.06N/m. Feedback gain was maintained high enough to pro-vide z-images with correct heights. Calibration of verticaland lateral distances of the AFM images was performed byusing a calibration grid CAL 3000/500-A (DME A/S, Her-lev, Denmark). All images were obtained with the samecantilever and under the same load and scan rates. Scanrates were approximately 100 µm/s and scan forces weremaintained below 5 nN. Image analysis was made withNIH - Image (NIH, Bethesda, Md., USA). All images wereacquired with 256 × 256 pixels and are shown as raw datafrom the AFM. No convolution matrix was applied to theimages; only contrast adjustments were done.

Electron Spectroscopic Diffraction (ESD)

The shells were washed three times in distilled water,mechanically grounded to powder, mixed with deionizedwater, captured with a formvar-covered electron micro-scope grid (200 mesh, Pelco International) by touching thegrid to the surface of a drop with the samples, and air dried.

R. Cruz et al.: FESEM and AFM of mollusk shell 13

The diffraction pattern obtained from the samples wasrecorded with the energy-filtering electron microscopeZeiss CEM 902 (Carl Zeiss) operating at 80 kV (energy-selecting slit: 20 eV; Reimer et al. 1990).

Results

The morphology and shell diagnosis of Micropilinaarntzi had been described previously (Warén and Hain1992); however, in the present study the FESEM operat-ing at low voltage and the AFM provided new informa-tion, especially about details of the shells at a nanomet-ric level. Under polarization light microscopy, the shellshowed an approximately circular projected contour, witha inhomogeneous contrast due to differences in local bir-refringence characteristics, which reflects the structuralorganization of the crystallites inside the shell (Fig. 1). Aclose view of the external surface shows that it is period-ically pitted by shallow depressions (Fig. 2, insert). In

FIG. 1 Light microscopy image of Micropilina arntzi shell viewedbetween crossed polarizers showing high contrast due to birefringentnature of the mineral.

FIG. 2 Field emission scanning electron microscopy micrograph ofexternal surface of the shell showing details of punctate sculpture (in-sert). Bar represents 100 µm in the insert.

14 Scanning Vol. 25, 1 (2003)

fractured shells it was possible to discern that they arecomposed by three layers. These layers differ in the ori-entation, morphology, and aggregation of the crystallites.The external surface (first layer) of the shell is composedby smooth structures (Fig. 3, white arrow). Just below thesurface (Fig. 3, asterisk), the second layer consists ofnacreous tablets. The boundaries of the nacreous stackedtablets present both curved and straight outlines (Fig. 4).The ESD obtained from macerated clusters of the shell ofMicropilina arntzi showed only the rings of aragonite(CaCO3) and spots of monocrystals (Fig. 4, insert). Theinternal side of the shell (third layer) near the peripheryexhibits thin sheet-like structures with parallel boundaries(Fig. 5). The angle between adjacent edges of each sheetis approximately 90°. Imaging by FESEM at higher mag-nification and low voltage (2 kV) showed that the sheet-like structures present a rough surface (Fig. 6). This sur-

face comprises small crystallites aligned in parallel to thelonger axis of each sheet.

When imaged at low voltage (2 to 5 kV), the middle re-gion of the inner side (third layer) of the shell displayed threearrangement patterns (Figs. 7–9). The first arrangement iscomposed by overlapping thin sheet-like structures with rec-tangular outlines (Fig. 7). The surfaces of the thin sheet-likestructures have very fine, concentric, and regularly spacedgrowth striations. The pattern of the concentric growth stri-ations is present in different overlapping sheet-like struc-tures. The second arrangement pattern is also constituted bysheet-like structures (Fig. 8); however, in this case each ofthe surfaces displays chevron-like striations regularly spacedon the surface. The third pattern found consists of thick su-perposed tablets (Fig. 9). The corners of the tablets areround and the tablet’s outlines present the same geometryas the growth striations seen at their surfaces.

FIG. 3 Field emission scanning electron microscopy image of frag-mented shell displaying the external surface (arrow) and internalnacreous layers (asterisk).

FIG. 4 Field emission scanning electron microscopy image of nacre-ous tablets showing both curved and straight outlines. Insert: Elec-tron spectroscopic diffraction pattern of macerated shell, showingrings of aragonite (CaCO3).

FIG. 5 Field emission scanning electron microscopy image of sheet-like structures showing rectangular profiles.

FIG. 6 High magnification and low voltage (2 kV) of the sheet-likestructures depicted in Figure 5 imaged at low voltage. Note the pres-ence of small crystallites on the surface.

R. Cruz et al.: FESEM and AFM of mollusk shell 15

The shell fragments of the internal side near the apex pre-sented high thickness and screw dislocations (Figs. 10,11), and stacked up structures (Fig. 12). These surfaces pre-sent rough texture and well-defined striations. Stackedsheets compose the thickness of the screw dislocationstructures (Fig. 11). The space between each sheet is par-tially filled by columns of crystallites oriented perpendic-ularly to the surface, and empty cavities (Fig. 11). Thestacked-up structures in this region display approximatelyhexagonal outlines (Fig. 12).

Atomic force images and profile plots of the sheet-likestructures depicted in Figure 9 revealed that the sheets arearranged in parallel (Fig. 13), and the step height of eachsheet is approximately 0.5 µm. At higher magnification,the surface of the structure examined by AFM displays arough texture (Fig. 14). The surface is irregular and showsrounded individualized features with an approximate heightof 50–100 nm.

Discussion

Most of the studies on the microstructure of molluskshells employed conventional SEM with tungsten (W) orlanthanum hexaboride (LaB6) electron emitters (Hede-gaard 1997, Mutvei 1978, Wilmot et al. 1992). Due to theinherent low brightness of these sources (Joy 1984), rela-tively high voltages (15–25 kV) are needed to ensureenough current for good imaging. The nonconductive cal-cium carbonate minerals of mollusk shells require the useof relatively thick (10–50 nm; e.g., Hedegaard 1997) con-ductive layers to avoid sample charging. These layers areoften very thick and obliterate the visualization of finesurface details of the shells surfaces.

The field emission electron gun of the FESEM providesa small beam probe diameter with a high-current densityand an exceptionally high brightness (Stokroos et al. 1995).Due to these particularities the FESEM makes possible the

FIG. 7 Sheet-like structures with rectangular outlines. Note the fineconcentric growth striations on the surface.

FIG. 8 Chevron-like growth striations belonging to stacked sheets.

FIG. 9 Several thick tablets superimposed on each other with con-centric growth striation on the surface.

FIG. 10 Field emission scanning electron microscopy image of acurled structure. Note the stacked sheets in the z-axis and rugose sur-face texture. Note the high quality of the image at the edge contours.

16 Scanning Vol. 25, 1 (2003)

observation of samples with low accelerating voltages(Goldstein et al. 1992, Stokroos et al. 1995). The use of alow accelerating voltage in association with an ultra-thinconductive layer on the specimen surface allowed theachievement of higher resolution (Goldstein et al. 1992).

The delicate growth striation lines and other fine detailsobserved in FESEM images of fragmented shell of Mi-cropilina arntzi were attained due to the low acceleratingvoltages (2–5 kV) employed and to the very thin conduc-tive layer (3 nm) used. We conclude that the employedtechniques contributed significantly to an improvement in

imaging the microstructure of Micropilina arntzi shell andmay contribute to be an important tool for the study of mol-lusks shell microstructure. Thus, it was possible to imagethe fine details of the surface without any etching or fur-ther cleaning with NaOCl or NaOH, as usually performedwhen conventional SEM is employed.

The use of higher voltages in conventional SEM with Wor LaB6 sources generates electron beams which penetratemore deeply into the specimen (Goldstein et al. 1992,Stokroos et al. 1995). The higher penetration produces anincrease in the brightness in the edges, resulting in loss of

FIG. 11 High magnification from a region depicted in Figure 10. Ob-serve the empty cavities and columns of crystallites growing in thez-axis.

FIG. 12 Stacked-up structures displaying an outline approximatelyhexagonal. Curled structures depicting both right- and left-handed ori-entations are seen.

FIG. 13 Atomic force microscopy image and profile plot measure-ment of the sheet-like structure. The measured region was a diagonalline extending from bottom left to the top right. It can be observed thatthe structures are parallel and present a step height about of 0.5 µm.

FIG. 14 Atomic force microscopy image at higher magnification ofthe sheet-like structure surface showing rounded particles with vari-able morphologies and dimensions. The white line delineates the mea-sured region.

microstructure resolution. This effect is dependent upon theaccelerating voltage. The use of low accelerating voltagesin this study permitted the observation of the stacked struc-tures and cavities in the border of the shell structures of Mi-cropilina arntzi. This approach may be an useful tool forthe visualization of fine details in nonconductive biomin-eralized structures.

The AFM provides measurement on X, Y, and particu-larly Z axis with high accuracy. When this technique iscombined with high-resolution SEM as in the case ofFESEM, we can attain information of the topography andheight of the samples. As a consequence of the depth offield and the orthogonal projection of the sample presentin SEM images, it is impossible to evaluate the correctheight of the sample just by looking at the image (Gold-stein et al. 1992). Height evaluation using SEM is per-formed by using stereoscopic projections; however, theresolution of the AFM in this case is more accurate.

The contribution of the AFM to measuring the stepheight of sheet-like structures seen in Figure 9 is unique(graphics in Fig. 13). The value found (0.5 µm), shows howfar the observer may be from the real topography of the ma-terial when trying to interpret the third dimension of thesample just by looking at SEM images (compare the mag-nification bar in Fig. 9 with the projection of the lateral sur-faces of the sheet-like structures). The use of AFM andFESEM to study similar regions of the same sample, as inthis work, shows the importance of so-called correlative mi-croscopy, especially in evaluating the third dimension am-plitudes at the nanometer scale (Neves et al. 1999).

The AFM images showed that some of the nacreoustablets (sheet-like structures) found in Micropilina arntzishell are arranged in parallel. Other structures (stacked-upstructures) presented slightly curved outlines. These dataindicate that the Micropilina arntzi, a primitive mollusk, iscapable of mineralizing several forms of the same mineral(aragonite).

The region presenting screw dislocation observed in theshell of Micropilina arntzi resembles those found in bio-mineralized structures from other invertebrate phyla. Thecalcitic skeleton biomineralized by the cyclostome bry-ozoans (Invertebrata:Bryozoa) described by Weedon andTaylor (1995) displays similar screw dislocations as thosefound in the aragonitic shells of Micropilina arntzi. Theseobservations indicate that distinct taxonomic groups mayproduce similar biomineralized structures of both poly-morphs of calcium carbonates minerals (aragonite and cal-cite).

Cleavage of shells from the class Gastropoda and Bi-valvia along aragonite tablet boundaries revealed nanoscalemineral islands (asperities) of about 30–100 nm in diam-eter, 10 nm in amplitude, separated by 60–120 nm, creat-ing a “peak and valley topography” (Wang et al. 2001).Nanoasperities at the aragonite tablet surfaces are the prin-cipal source of the shear resistance between adjacenttablets, accounting for the stress behavior at which the in-elastic deformation proceeds in mechanical assays de-

R. Cruz et al.: FESEM and AFM of mollusk shell 17

signed to study deformation mechanics in nacre. In Figure14, the roughness of the tablet’s surface of the third layerof Micropilina arntzi shell is presented. The diameters ofparticles present in this surface are in the range of those ob-served by Wang et al. (2001). Possibly, the particles ob-served in Micropilina arntzi represent nanoasperities sim-ilar to those found in Gastropoda and Bivalvia shells.

The shell microstructure examined in this study differsfrom other species of Monoplacophora previously ana-lyzed (McLean 1979, Warén and Gofas 1996). The shellsof the monoplacophoran family Neopilinidae, described byMcLean (1979) and Warén and Gofas (1996), present pris-matic structures with a hexagonal surface outline. We didnot find similar structures in Micropilina arntzi (Mi-cropilinidae). The shell microstructures of the Monopla-cophora species present different patterns, and this fact mayhave a taxonomic implication in the systematic studies ofthe group.

Acknowledgment

The authors are indebted to Dr. Anders Wáren (SwedishMuseum of Natural History) for providing the material de-scribed in this study.

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