Earth’s rock record holds great potential for decoding the origin and early diversification of life on our planet. However, the interpretation of the Precambrian (older than ~541 million years ago) fossil record is fraught with difficulties. These include: the fragmentary nature of the sedimentary rock record with large periods of time unrepresented and certain habitats under-represented; and the nature of the organisms, being microscopic, morphologically simple and often only subtly different from co-occurring non-biological organic material.

Distinguishing between true signs of life and abiotic artefacts requires analytical techniques with excellent spatial resolution in two and three dimensions, in order to accurately analyse key features of putative cells such as cell wall ultrastructure, biochemistry, and interaction of cell walls with the minerals that have fossilised them [1]. Likewise, distinguishing different grades of life (for example, simple prokaryotes versus more complex eukaryotes) requires similar techniques, in order to identify putative multi-cellularity and specific types of cell contents and cell wall architecture. 

We here demonstrate how a protocol combining focused ion beam (FIB) milling, SEM, TEM and nano-scale secondary ion mass spectrometry (SIMS) can reveal unprecedented  nanometer to micrometer scale details of Precambrian fossilised organisms, providing more robust biosignatures for both prokaryotes and eukaryotes for future studies on Earth or other planets. 

FIB milling was used to prepare ultrathin (c. 100 nm) wafers from standard geological thin sections for TEM analysis, plus slightly thicker wafers (c. 150-200 nm) that could be used for both TEM and NanoSIMS analysis. The latter meant that both TEM and NanoSIMS data could be collected from a single candidate microfossil: TEM data included ChemiSTEM elemental mapping of major elements, STEM-EELS analysis of the bonding and structure of organic material, and electron diffraction to identify mineral phases; NanoSIMS data included targeted analysis of trace elements in organic material (e.g., N, S, P) and in the fossilising mineral phases. Analysis of FIB-milled wafers counteracts the problems previously associated with surface analysis techniques such as NanoSIMS (i.e. surface contamination and polishing effects). FIB-milling was also combined with SEM imaging (3D slice and view) in order to obtain accurate 3D visualisations of candidate microfossils.

Data will be presented from three geological formations that play an important role in our understanding of the origin and evolution of early life on Earth: 1, The 1878 Ma Gunflint Formation of Canada, containing an iconic suite of diverse microfossils used as a benchmark for high quality preservation of early life in marine environments [2]; 2, The 1000 Ma Torridon Group of northwest Scotland (Fig. 1) that is renowned for exceptional three-dimensional preservation of both prokaryotes and eukaryotes in phosphate and clay minerals in a terrestrial (lake) setting [3]; 3, The 850 Ma Bitter Springs Formation of central Australia that shows exquisite microfossil preservation (including putative cell contents) in micro-quartz [4].

References cited

[1] D. Wacey et al. (2011) Nature Geosci. 4, 698-702.

[2] D. Wacey et al. (2013) PNAS 110, 8020-8024.

[3] P. Strother et al. (2011) Nature 473, 505-509.

[4] J.W. Schopf (1968) J. Palaeontology 42, 651-688.

Acknowledgements

DW acknowledges funding from the Australian Research Council and European Commission. KE is supported by an Australian Postgraduate Award and a UWA Top-up Scholarship. We acknowledge the facilities, scientific and technical assistance of the AMMRF at UWA, a facility funded by the University, State and Commonwealth Governments. Paul Strother is thanked for provision of the Torridon specimens.

Figures:

Figure 1. (a) Optical photomicrograph of a well-preserved microfossil with internal cell contents from the 1000 Ma Torridon Group of northwest Scotland. (b) FIB-SEM image through the microfossil shown in (a), showing cell wall and cell contents in black and at least two different types of fossilizing minerals (varying shades of grey). (c) FIB-milled TEM wafer through the microfossil shown in (a). (d) Composite and false-coloured ChemiSTEM elemental map of carbon from the boxed area of the wafer shown in (c). This confirms the organic nature of the cell wall and cell contents.

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