atomic force microscopy imaging of fragments from the martian meteorite alh84001

SHORT COMMUNICATION

Atomic force microscopy imaging of fragments from theMartian meteorite ALH84001

A. STEELE,* D. GODDARD,† I. B. BEECH,* R. C. TAPPER,* D. STAPLETON‡ & J. R. SMITH**School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building,White Swan Road, Portsmouth PO1 2DT, U.K.†BNFL, Company Research Laboratory, Springfields Site, Preston, U.K.‡Xyratex Engineering Laboratory, Xyratex, Havant, Hampshire, U.K.

Key words. Atomic force microscopy, AFM, Martian meteorite ALH84001,magnetite, nanofossils, scanning electron microscopy.

Summary

A combination of scanning electron microscopy (SEM) andenvironmental scanning electron microscopy (ESEM) tech-niques, as well as atomic force microscopy (AFM) methodshas been used to study fragments of the Martian meteoriteALH84001. Images of the same areas on the meteoritewere obtained prior to and following gold/palladiumcoating by mapping the surface of the fragment usingESEM coupled with energy-dispersive X-ray analysis. View-ing of the fragments demonstrated the presence ofstructures, previously described as nanofossils by McKayet al. (Search for past life on Mars – possible relic biogenicactivity in martian meteorite ALH84001. Science, 1996, pp.924–930) of NASA who used SEM imaging of gold-coatedmeteorite samples. Careful imaging of the fragmentsrevealed that the observed structures were not an artefactintroduced by the coating procedure.

Introduction

Since the announcement by D. McKay et al. in August 1996of the possible existence of primitive fossilized organismswithin a known Martian meteorite, ALH84001 (McKay etal., 1996), the scientific community has been divided. Thearguments supporting and disclaiming the evidence gath-ered by Dr McKay and his colleagues have raged loudly andpublicly (Bradley et al., 1996, 1997; McSween, 1996;Thomas-Keprta et al., 1997; Golden et al., 1997 and otherrelevant papers presented at the 28th Lunar and PlanetaryScience Conference, 17–21 March 1997, Houston).

Scanning electron microscopy (SEM) images of structureslabelled as possible nanofossils, detected within carbonateconcretions, have been presented as part of the evidence forthe existence of primitive life on Mars. However, theinterpretation of the images has been disputed, with themajor critics postulating that these structures are eitherartefacts generated by the conductive Au/Pd coatingrequired to perform SEM analysis, or mineral in origin(Gibbs & Powell, 1996).

The present study aimed to further elucidate the natureof reported nanostructures. In order to avoid the introduc-tion of artefacts due to specimen preparation, microscopicaltechniques free of sample pretreatment procedures, such asatomic force microscopy (AFM) imaging combined withenvironmental SEM (ESEM) examination, have beenemployed.

AFM allows observation of nonconducting materials atthe atomic level (Goh & Markiewicz, 1992; Haggerty &Lenhoff, 1993). In the area of biological imaging, the highresolution and three-dimensional capability of AFM, com-bined with minimal sample preparation, has been exploitedto investigate proteins, nucleic acids, cells and cellularstructures (Gould et al., 1990; Butt et al., 1990; Haggerty &Lenhoff, 1993; Southam et al., 1993; Steele et al., 1994;Muller et al., 1996). As the topographical data are collectedby scanning the sharp tip across the surface of a specimen,the AFM images generated are subject to artefacts (Edstrom,1990; Butt et al., 1993; Haggerty & Lenhoff, 1993). Theappearance of artefacts in AFM has been studied in detailand can be compensated for in two ways: either using AFMimage analysis software, or employing another techniquesuch as SEM to verify the images obtained with AFM (Steeleet al., 1995). Direct comparison of the same area on the

Journal of Microscopy, Vol. 189, Pt 1, January 1998, pp. 2–7.Received 20 October 1997; accepted 18 November 1997

2 q 1998 The Royal Microscopical Society

Correspondence to: Dr R. C. Tapper

Current address of A. Steele: Mailcode SN2, Building 31, NASA Johnson Space

Center, Houston, TX 77058, U.S.A. Email: [email protected]

sample characterized by a combination of these twotechniques leads to a clearer, artefact-free image.

In the present study, a combination of the imagingtechniques has been applied to analyse exactly the sameareas of the meteorite fragments, both prior to and aftergold/palladium treatment. The investigation aimed todetermine whether the structures reported as nanofossilswere artefacts introduced following sample coating.

Materials and methods

Chips of ALH84001 (0·3–0·5 mm in diameter), supplied byDr D. S. McKay of NASA Johnson Space Center and Dr M.Grady of the Natural History Museum, London, weremounted on 10-mm-diameter stainless steel stubs usingcarbon tape. Each sample of ALH84001 was opticallyimaged to identify the bounds of any carbonate globulespresent on the surface. Environmental SEM (ESEM) (PhilipsElectroScan 2020) fitted with light element energy-disper-sive X-ray analysis (EDX, Oxford Instruments) was thenemployed to image and map the distribution of elementsacross the entire surface of each sample. The advantage ofusing ESEM was that samples required no pretreatment.The low-power images (×2000) of the surface werecombined to form a comprehensive map of the surface, onwhich areas of interest, both part of and removed from thecarbonate globules, were identified.

A Nanoscope III atomic force microscope (Digital Instru-ments, U.S.A.), operating in intermittent contact mode(Wiegraße et al., 1991; Bustamante et al., 1994), was usedto image the identified areas of the sample. The microscopeused standard etched Si3N4 cantilevers resonating at

300 kHz, with a spring constant of between 20 and60 N m–1. After initial imaging of the surface, specimenswere coated with gold/palladium as described by McKay etal. (1996). The ESEM mapping of the surface allowed themicroscope to re-image the same areas as prior to coating.Thus comparisons of the change in the sample surfacetopography due to coating could be carried out. Finally, SEMimaging of the entire surface of the fragments was carriedout and any interesting features observed were furtherimaged using AFM.

As a control to this procedure, 1-cm2 mica wafers wereimaged with AFM after Au/Pd coating using a magnetronsputter coater (EMITECH K550, U.K.). The coating wascarried out to mimic the conditions adopted by McKay et al.(1996) but carried out over 10, 25 and 60 s and at 25 and45 mm target/sample distances. Average grain size analysiswas conducted for three representative images per para-meter using the AFM image analysis software (Nanoscope,Digital Instruments, U.S.A.). A paired Student t-test atP ¼ 0·05 was used for statistical assessment of the results.

Results

ESEM imaging (40 mm × 40 mm) of the rim of a carbonateglobule of a fragment of ALH84001 supplied by McKay et al.(1996) (Fig. 1A) and EDX mapping of Fe, Mg and S in thisarea are presented in Fig. 1(B–D), respectively. The rim ofthe carbonate globule runs from the top right to the bottomleft of the image. To the right of this rim is theorthopyroxenite, the dominant igneous mineral inALH84001; to the left is the carbonate globule. Thepresence of the phases revealed in Fig. 1(B–D) demonstratedthat there is an inner and an outer iron/sulphur-rich rimsurrounding a magnesium-rich area. This finding is in goodagreement with the published literature (McKay et al.,1996; Harvey & McSween, 1996). EDX analysis was usedonly for qualitative observations because of the irregularnature of the surface.

AFM imaging of areas across the rim of carbonateglobules, shown in Fig. 2(A), reveals the presence of small(100–200 nm) ovoid particles. The appearance of thesestructures was consistent for several sites analysed withinthe Fe/S-rich area of the rim of the carbonate concretions.Similar features were also seen across the surface of thesamples, regardless of the presence of the carbonateglobules, as demonstrated in Fig. 2(B).

No discernible difference in the surface appearance of thesample prior to and after coating with Au/Pd could be seenwhen imaging the samples at a scan size of 4 mm (Fig. 3A,B,respectively). In these images elongated structures appear tobe in distinct lines which run from the bottom left to the topright of the image. These structures seem to be amalgama-tions of small spherical particles (100–200 nm) and occurover the whole surface of the samples.

q 1998 The Royal Microscopical Society, Journal of Microscopy, 189, 2–7

Fig. 1. (A) An SEM image of the rim of a carbonate globule. (B–D)EDX mapping demonstrating the distribution of Fe, Mg and S,respectively, in the same area as (A). In these images the lighterthe colour, the higher the concentration of elements.

AFM OF MARTIAN METEORITE ALH84001 3

The analysis of the grain size of the Au/Pd coatingsputtered on mica reveals that the average particle size atthe 20–30 s time frame used by McKay et al. (1996) iswithin the 6–8 nm range, although this is dependent uponthe sample-to-target distance (Fig. 4). Data collected onhigher magnification AFM observation of ALH84001fragments prior to and after Au/Pd coating (Fig. 5A andB, respectively) demonstrates that there is an increase insurface roughness after coating. The surface appears to becovered with small Au/Pd nucleation points, with anaverage diameter of 6–7 nm, which is consistent with thedata collected from the coating of mica (Fig. 4). Thisappearance was constant across the surface of the speci-mens regardless of the presence of carbonate concretions.

The elongated segmented structures imaged by AFM afterAu/Pd coating (Fig. 6A) support the claims of McKay et al.(1996) that such features are present on the surface ofALH84001. SEM imaging of a fragment after Au/Pd coatinginitially identified these structures in an area remote from a

carbonate globule. The same zone was then re-imaged withAFM. Figures 6(A) and (B) are images obtained using AFMin phase mode. In phase imaging, the phase shift betweenthe excitation applied to the cantilever and its response ismeasured. Such a mode of imaging has been shown to besensitive to the material properties of the sample to beinvestigated. However, in the present work, AFM phasemode was useful as a method of contrast enhancement,sharpening fine features normally obscured by larger scaletopography. Although the segmented features only appearto be a part of the surface in the AFM images, SEMobservations verify that these structures do in fact protrudefrom the surface of the fragment.

Discussion

The atomic force microscope demonstrated an excellentcapability for the reproducible imaging of surfaces as roughas ALH84001 when located in areas of interest identified

Fig. 2. (A) A 3-D surface plot AFM image of the Fe/S rim. (B) A 3-D surface plot of an area removed from any carbonate globules.

Fig. 3. (A) A 2-D AFM image of an area on the carbonate globule (scan size 4 mm × 4 mm) before gold/palladium coating. (B) The same areaonly after Au/Pd coating.

4 A. ST EELE ET AL.


using ESEM mapping. EDX analysis of the surface (Fig. 1B–D) facilitated AFM imaging of the different phases of thecarbonate globules at nanometre resolution. The surfacefeatures in these phases appeared to differ. In the Fe/S-richregions there were a considerable number of roundedstructures 100–200 nm in diameter protruding from thesurface (Fig. 2A). It is proposed that these are either FeS ormagnetite crystals (McKay et al., 1996; Harvey & McSween,1996; Golden et al., 1997). Within areas of the carbonateglobules these rounded extrusions appeared to clumptogether in distinct lines across the surface (Fig. 3A); thiscould possibly be due to weathering.

The relocation of areas on the sample enabled acombination of imaging techniques to be utilized and

thus allowed a quantitative assessment of the effects ofsample coating prior to analysis. Direct comparison ofimages before and after Au/Pd coating (Figs 3 and 5) andgrain-size analysis of the mica control surfaces (Fig. 4)demonstrated that the coating is only responsible forgenerating artefacts in the 6–8 nm size range. This is ingood agreement with available literature. Therefore thestructures imaged by McKay et al. (1996) are not a resultof the sample preparation procedure. Furthermore, bothovoid and segmented, rod-shaped structures are present,not only as part of the carbonate globules, but aredistributed over the entire surface of the imaged fragments(Fig. 2B). If indeed these structures are nanofossils, asproposed by McKay et al. (1996), the fossilization may have


Fig. 4. The average grain size (nm) of gold/palladium sputter-coated on to the surfaceof mica. Samples were sputtered for 10,25 and 60 s at target–sample separationdistances of 25 and 45 mm.

Fig. 5. AFM images showing 400-nm areas of the surface of a carbonate globule prior to (A) and after (B) Au/Pd coating.


occurred independently of the formation of the carbonateconcretions.

The elongated, segmented structures (Fig. 6A,B) imagedusing AFM seem to resemble features reported by McKay etal. (1996). However, it is still a matter of speculationwhether these forms are indeed nanofossils or of crystallineorigin. Based on their orientation and size range, structuressuch as the ones observed on the Martian sample arereported for both the possible nanofossils as well asbiogenically and inorganically produced magnetite crystals(DeLong et al., 1993; Hansma & Hoh, 1994; Farina et al.,1994; Golden et al., 1997). When the structures are studiedunder higher magnification (Fig. 6B), individual nonsmoothsegments can be distinguished. Two obvious types of featurecan be seen: smooth, ripple-like waves on the surface, some20–50 nm long, and round protrusions in the 6–8 nmrange. The latter strongly resemble the nucleation points ofthe Au/Pd coating shown in Fig. 5(B). As to the nature ofthese features, research using techniques applied to frag-ments of ALH84001 to investigate terrestrial microfossils,basaltic analogues to ALH84001, magnetite crystals andother meteorites, is currently in progress. This ongoingstudy aims to compare the structures associated with theMartian meteorite fragments with known terrestrial analo-gues.

New developments in high-resolution microscopy pro-mise exciting opportunities for the study of geologicalspecimens. However, great care must be taken to avoidmisinterpretation of the imaged features. Only throughconsiderable effort to understand the structure of materialsoriginating from our own planet at the nanoscale level canaccurate comparison with samples of extraterrestrial originbe made.

Acknowledgments

We thank BNFL plc and Xyratex for backing this project byproviding instrumentation free of charge. Gratitude is alsoexpressed to the following: T. C. Crabb, M. Jones and D.Martill at the University of Portsmouth, M. Grady, E. K.Gibson, D. S. McKay, K. Thomas-Keprta, S. Worthington, G.Grimes, K., J. Toporski, C. Pillinger and I. P. Wright.

References

Bradley, J.P., Harvey, R.P. & McSween, H.Y. (1996) Magnetitewhiskers and platelets in the ALH84001 Martian meteorite:evidence of vapor phase growth. Geochim. Cosmochim. Acta, 60,5149–5155.

Bradley, J.P., McSween, H.Y. & Harvey, R.P. (1997) Epitaxial growthof single domain magnetite in Martian meteorite Allan Hills84001. Meteorit. Planet. Sci. 32, A20.

Bustamante, C., Erie, D.A. & Keller, D. (1994) Biochemical andstructural applications of scanning force microscopy. Curr. Opin.Struct. Biol. 4, 750–760.

Butt, H.J., Prater, C.B. & Hansma, P.K. (1990) Imaging cells withAFM. J. Vacuum Tech. 2, 1193–1196.

Butt, H.J., Siedle, P., Seifert, K., Fendler, K., Seeger, T., Bamberg, E.,Weisenhorn, A.L., Goldie, K. & Engel, A. (1993) Scan speed limitin atomic force microscopy. J. Microsc. 169, 75–84.

Delong, E.F., Frankel, R.B. & Bazylinski, D.A. (1993) Multipleevolutionary origins of magnetotaxis in bacteria. Science, 259,803–806.

Edstrom, R.D. (1990) Viewing molecules with STM and AFM.Atomic Force Microsc. 4, 3144–3151.

Farina, M., Kachar, B., Lins, U., Broderick, R. & Debarros, H.L.(1994) The observation of large magnetite (Fe3O4) crystals frommagnetotactic bacteria by electron and atomic-force microscopy.J. Microsc. 173, 1–8.

Fig. 6. AFM images of a segmented elongated rod structure in an area on an Au/Pd-coated ALH84001 fragment away from a carbonateglobule. (A) A 400-nm scan size image of the structure. (B) A 200-nm square image across an individual segment of the structure (A), show-ing distinct surface features.

6 A. ST EELE ET AL.


Gibbs, W.W. & Powell, C.S. (1996) Bugs in the data – thecontroversy over martian life is just beginning. Sci. Am. 275, 20.

Goh, C.M. & Markiewicz, P. (1992) The atomic force microscope.Chem. Ind. 18, 687–691.

Golden, D.C., Thomas-Keprta, K.L., Mckay, D.S., Wentworth, S.J.,Vali, H. & Ming, D.W. (1997) Size distribution of magnetite incarbonate globules of ALH 84001 martian meteorite. Proc. 28thLunar and Planetary Science Conference, Houston. Lunar andPlanetary Institute, NASA Johnson Space Center, Houston.

Gould, S.A.C., Drake, B., Prater, C.B., Weisenhorn, A.L., Manne, S.,Hansma, H.G. & Hansma, P.K. (1990) From atoms to integratedcircuit chips, blood cells and bacteria with the AFM. J. VacuumTech. 1, 369–373.

Haggerty, L. & Lenhoff, A.M. (1993) STM and AFM inbiotechnology. Biotechnol. Prog. 9, 1–11.

Hansma, H.G. & Hoh, J.H. (1994) Biomolecular imaging with theatomic force microscope. Annu. Rev. Biophys. Biomol. Struct. 23,115–239.

Harvey, R.P. & McSween, H.Y. Jr (1996) A possible high-temperature origin for the carbonates in the martian meteoriteALH84001. Nature, 382, 49–51.

McKay, D.S., Gibson, E.K., Thomaskeprta, K.L., Vali, H., Romanek,C.S., Clemett, S.J., Chillier, X.D.F., Maechling, C.R. & Zare, R.N.(1996) Search for past life on Mars – possible relic biogenicactivity in martian meteorite ALH84001. Science, 273, 924–930.

McSween, H.Y. (1996) Evidence for ancient-life in a martian

meteorite (exclamation or questionable). Meteorit. Planet. Sci. 31,691–692.

Muller, D.J., Baumeister, W. & Engel, A. (1996) Conformationalchange of the hexagonally packed intermediate layer ofDeinococcus radiodurans monitored by atomic-force microscopy.J. Bacteriol. 178, 3025–3030.

Southam, G., Firtel, M., Blackford, B.L., Jericho, M.H., Xu, W.,Mulhern, P.J. & Beveridge, T.J. (1993) Transmission electron-microscopy, scanning tunneling microscopy, and atomic forcemicroscopy of the cell-envelope layers of the archaeobacteriumMethanospirillum hungatei gp1. J. Bacteriol. 175, 1946–1955.

Steele, A., Goddard, D.T. & Beech, I.B. (1994) An atomic forcemicroscopy study of the biodeterioration of stainless steel in thepresence of bacterial biofilms. Int. Biodet. Biodeg. 33, 35–46.

Steele, A., Goddard, D.T. & Beech, I.B. (1995) A guide tovisualisation of single bacterial cells, biofilms and corrosiondamage of stainless steel using the technique of atomic forcemicroscopy. Corrosion/95 (ed. by P. Angell et al.), Paper 73.National Association of Corrosion Engineers, Houston, Texas.

Thomas-Keprta, K.L., Wentworth, S.J., McKay, D.S., Taunton, A.E.,Allen, C.C., Romanek, C.S. & Gibson, E.K. (1997) Subsurfaceterrestrial microfossils from Columbia river basalt samples:analogs of features in Martian meteorite Allan Hills 84001?Meteorit. Planet. Sci. 32, SS, A128–A129.

Wiegraße, W., Nonnenmacher, M., Guckenberger, R. & Wolter, O.(1991) Atomic force microscopy of a hydrated bacterial surfaceprotein. J. Microsc. 163, 79–84.



atomic force microscopy imaging of fragments from the martian meteorite alh84001

Documents