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Generation of living cell arrays for atomic force microscopy studies

Nature Protocols volume 10, pages 199–204 (2015)Cite this article

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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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Figure 1: Schematics of the protocol for living cell immobilization.
Figure 2: Characterization of the PDMS stamps obtained.
Figure 3: Imaging of a C. albicans cell array.
Figure 4: Multiparametric imaging of a C. albicans array.

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References

  1. Binnig, G., Quate, C.F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–934 (1986).

    Article  CAS  Google Scholar 

  2. Gerber, C. & Lang, H.P. How the doors to the nanoworld were opened. Nat. Nanotechnol. 1, 3–5 (2006).

    Article  CAS  Google Scholar 

  3. Louise Meyer, R. et al. Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy 110, 1349–1357 (2010).

    Article  CAS  Google Scholar 

  4. Doktycz, M.J. et al. AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces. Ultramicroscopy 97, 209–216 (2003).

    Article  CAS  Google Scholar 

  5. Kasas, S. & Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995).

    Article  CAS  Google Scholar 

  6. Kailas, L. et al. Immobilizing live bacteria for AFM imaging of cellular processes. Ultramicroscopy 109, 775–780 (2009).

    Article  CAS  Google Scholar 

  7. Francius, G., Domenech, O., Mingeot-Leclercq, M.P. & Dufrêne, Y.F. Direct observation of Staphylococcus aureus cell wall digestion by lysostaphin. J. Bacteriol. 190, 7904–7909 (2008).

    Article  CAS  Google Scholar 

  8. Alsteens, D. et al. Structure, cell wall elasticity and polysaccharide properties of living yeasts cells, as probed by AFM. Nanotechnology 19, 384005 (2008).

    Article  Google Scholar 

  9. Dague, E., Alsteens, D., Latgé, J.-P. & Dufrêne, Y.F. High-resolution cell surface dynamics of germinating Aspergillus fumigatus conidia. Biophys. J. 94, 656–660 (2008).

    Article  CAS  Google Scholar 

  10. Gilbert, Y. et al. Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction. Nano Lett. 7, 796–801 (2007).

    Article  CAS  Google Scholar 

  11. Dague, E. et al. Assembly of live micro-organisms on microstructured PDMS stamps by convective/capillary deposition for AFM bio-experiments. Nanotechnology 22, 395102 (2011).

    Article  CAS  Google Scholar 

  12. Francois, J.M. et al. Use of atomic force microscopy (AFM) to explore cell wall properties and response to stress in the yeast Saccharomyces cerevisiae. Curr. Genet. 59, 187–196 (2013).

    Article  CAS  Google Scholar 

  13. Pillet, F., Chopinet, L., Formosa, C. & Dague, É. Atomic force microscopy and pharmacology: from microbiology to cancerology. Biochim. Biophys. Acta 1840, 1028–1050 (2014).

    Article  CAS  Google Scholar 

  14. Chopinet, L., Formosa, C., Rols, M.P., Duval, R.E. & Dague, E. Imaging living cells surface and quantifying its properties at high resolution using AFM in QITM mode. Micron 48, 26–33 (2013).

    Article  CAS  Google Scholar 

  15. Formosa, C. et al. Nanoscale effects of Caspofungin against two yeast species, Saccharomyces cerevisiae and Candida albicans. Antimicrob. Agents Chemother. 57, 3498–3506 (2013).

    Article  CAS  Google Scholar 

  16. Pillet, F. et al. Uncovering by atomic force microscopy of an original circular structure at the yeast cell surface in response to heat shock. BMC Biol. 12, 6 (2014).

    Article  Google Scholar 

  17. Beauvais, A. et al. Deletion of the α-(1,3)-glucan synthase genes induces a restructuring of the conidial cell wall responsible for the avirulence of Aspergillus fumigatus. PLoS Pathog. 9, e1003716 (2013).

    Article  Google Scholar 

  18. Dufrêne, Y.F., Martínez-Martín, D., Medalsy, I., Alsteens, D. & Müller, D.J. Multiparametric imaging of biological systems by force-distance curve-based AFM. Nat. Methods 10, 847–854 (2013).

    Article  Google Scholar 

  19. Dufrêne, Y.F. Atomic force microscopy and chemical force microscopy of microbial cells. Nat. Protoc. 3, 1132–1138 (2008).

    Article  Google Scholar 

  20. Francius, G. et al. Stretching polysaccharides on live cells using single-molecule force spectroscopy. Nat. Protoc. 4, 939–946 (2009).

    Article  CAS  Google Scholar 

  21. Beaussart, A. et al. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 9, 1049–1055 (2014).

    Article  CAS  Google Scholar 

  22. Roduit, C. et al. OpenFovea: open-source AFM data processing software. Nat. Methods 9, 774–775 (2012).

    Article  CAS  Google Scholar 

  23. Cai, D.K., Neyer, A., Kuckuk, R. & Heise, H.M. Optical absorption in transparent PDMS materials applied for multimode waveguides fabrication. Opt. Mater. 30, 1157–1161 (2008).

    Article  CAS  Google Scholar 

  24. Chabinyc, M.L. et al. An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications. Anal. Chem. 73, 4491–4498 (2001).

    Article  CAS  Google Scholar 

  25. Liu, S. & Wang, Y. Application of AFM in microbiology: a review. Scanning 32, 61–73 (2010).

    Article  CAS  Google Scholar 

  26. JPK Instruments. QITM mode-quantitative imaging with the Nanowizard 3 AFM. http://usa.jpk.com/index.download.419baba450fa6c06a245970866047614.

  27. Formosa, C. et al. Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy. Nanomed. Nanotechnol. Biol. Med. 10.1016/j.nano.2014.07.008 (4 August 2014).

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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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Authors and Affiliations

  1. Centre Nationale de la Recherche Scientifique (CNRS), Laboratoire d'Analyse et d'Architecture des Systèmes (LAAS), Toulouse, France

    Cécile Formosa, Marion Schiavone & Etienne Dague

  2. Université de Toulouse,

    Cécile Formosa, Flavien Pillet, Marion Schiavone, Laurence Ressier & Etienne Dague

  3. LAAS, Institut des Technologies Avancées en Sciences du Vivant (ITAV), Institute de Pharmacologie et de Biologie Structurale (IPBS), Toulouse, France

    Cécile Formosa, Flavien Pillet, Marion Schiavone, Laurence Ressier & Etienne Dague

  4. CNRS, Unité Mixte de Recherche (UMR) 7565, Laboratoire Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Vandœuvre-lès-Nancy, France.,

    Cécile Formosa & Raphaël E Duval

  5. Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France

    Cécile Formosa & Raphaël E Duval

  6. CNRS, IPBS, UMR 5089, Toulouse, France

    Flavien Pillet

  7. L'Institut National de la Recherche Agronomie (INRA), UMR 972 Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Toulouse, France

    Marion Schiavone

  8. ABC Platform, Nancy, France

    Raphaël E Duval

  9. Université de Toulouse, Laboratoire de Physique et Chimie de Nano-Objets (LPCNO), L'Institut National des Sciences Appliquées (INSA)-CNRS-Université Toulouse III-Paul Sabatier, Toulouse, France

    Laurence Ressier

Authors
  1. Cécile Formosa
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  2. Flavien Pillet
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  4. Raphaël E Duval
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  5. Laurence Ressier
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  6. Etienne Dague
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Contributions

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.

Corresponding author

Correspondence to Etienne Dague.

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The authors declare no competing financial interests.

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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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