Abstract
Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.
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Acknowledgements
We thank Techniques et Equipements Appliqués à la Microélectronique (TEAM) engineers and especially A. Laborde for their technical support in silicon master fabrication. We thank V. Beges for artwork on Figure 1. This work was supported by an Agence Nationale de la Recherche (ANR) young scientist program (AFMYST project ANR-11-JSV5-001-01 no. SD 30024331) to E.D. E.D. is a researcher at the Centre National de Recherche Scientifique. C.F. and M.S. are, respectively, supported by a grant from 'Direction Générale de l'Armement' and by funding from Lallemand.
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E.D. and L.R. developed the concept and designed the experiments; E.D., L.R., R.E.D. and C.F. conceived and designed the experiments and wrote the article. E.D., C.F., F.P. and M.S. made the experimental work and the data analysis work; C.F., F.P. and M.S. worked on the experimental protocol; and all authors discussed the results and commented on the manuscript.
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Example CleWin file of a micropattern for the silicon master. (ZIP 320 kb)
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Formosa, C., Pillet, F., Schiavone, M. et al. Generation of living cell arrays for atomic force microscopy studies. Nat Protoc 10, 199–204 (2015). https://doi.org/10.1038/nprot.2015.004
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DOI: https://doi.org/10.1038/nprot.2015.004
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