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Generation of living cell arrays for atomic force microscopy studies Cécile Formosa1–4, Flavien Pillet2,5, Marion Schiavone1,2,6, Raphaël E Duval3,4,7, Laurence Ressier2,8 & Etienne Dague1,2 1Centre Nationale de la Recherche Scientifique (CNRS), Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Toulouse, France. 2Université de Toulouse;
LAAS, Institut des Technologies Avancées en Sciences du Vivant (ITAV), Institute de Pharmacologie et de Biologie Structurale (IPBS), Toulouse, France. 3CNRS, Unité Mixte de Recherche (UMR) 7565, Laboratoire Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Vandœuvre-lès-Nancy, France. 4Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France. 5CNRS, IPBS, UMR 5089, Toulouse, France. 6L’Institut National de la Recherche Agronomie (INRA), UMR 972 Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Toulouse, France. 7ABC Platform, Nancy, France. 8Université 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. Correspondence should be addressed to E.D. ([email protected]).
© 2014 Nature America, Inc. All rights reserved.
Published online 31 December 2014; doi:10.1038/nprot.2015.004
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.
INTRODUCTION Since its first development in 1986 (ref. 1), AFM has developed into a powerful tool that has opened the doors to the nanoworld2. AFM is particularly well suited for biology, as it allows multiple characterizations (topography, mechanical and adhesive properties) of living cells in their physiological environments. However, a prerequisite for such AFM experiments is the immobilization of the biological samples probed. However, this crucial step is often a challenge, as samples have to be immobilized individually and firmly enough to withstand the lateral forces exerted by the AFM tip, but without altering their cellular integrity. Immobilization of cells for AFM experiments Several techniques have been used to immobilize living cells. Living cells such as microorganisms can be chemically fixed on a solid substrate using glutaraldehyde or (3-aminopropyl)triethox ysilane (APTES)3, or they can be immobilized on gelatin-coated surfaces4. However, these techniques can, respectively, modify the interface of the biological sample, or they can pollute the AFM tips, leading to artifacts. Another strategy is to trap roundshaped cells such as bacterial cocci and yeast cells in the pores of polycarbonate membranes by filtration5 or in lithographically patterned substrates by gentle drying6. The filtration technique has been widely used over recent years7–10, although it is timeconsuming and cells can be exposed to mechanical forces when trapped in the pores. We therefore developed a new and versatile strategy in 2011 (refs. 11–13), which consists of immobilizing round single living cells in microstructured PDMS stamps by convective/capillary deposition. We have demonstrated that this generic protocol can be used to immobilize different types of round cells, ranging from small cocci bacteria to yeasts cells and even algae, by tuning the geometry of the PDMS stamp patterns (Supplementary Data).
This approach was also exploited to immobilize yeast cells of different species, Saccharomyces cerevisiae and Candida albicans14–16, as well as spores of Aspergillus fumigatus17. Statistical significance of results obtained from AFM: the new challenge Statistical analysis of results is desirable. As AFM is a tool adapted for single-cell analysis, this technology requires the analysis of multiple cells in order to achieve statistical confidence. This cannot be performed using techniques such as immobilization in a pore filter, as the deposition of the cells on the surface is random and the rate of filled pores is low and not controlled. By using our method of immobilization, it is possible to generate arrays of cells; therefore, AFM results on an array of 100 cells can be performed using different AFM modes, such as multiparametric imaging, chemical force microscopy, single-molecule force spectroscopy and single-cell force spectroscopy14,18–21. Such setups are effectively ‘lab chips’ for AFM analysis. With the progress made in AFM data processing software22, it is thus possible to take all the data acquired to analyze in a reasonable time period, so as to generate relevant and statistically significant results for biological studies. Overview of the procedure Immobilization of living cells in PDMS stamps involves three sequential stages. The first stage is the generation of a glass and chromium mask that harbors microstructured patterns, and the transfer of these patterns onto a silicon wafer. The second stage consists of the preparation of a corresponding PDMS stamp. Finally, the third stage is the assembly of the living cells into the PDMS microstructured stamps. The generation of a silicon master is achieved by photolithography, followed by pattern transfer nature protocols | VOL.10 NO.1 | 2015 | 199
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© 2014 Nature America, Inc. All rights reserved.
Figure 1 | Schematics of the protocol for living cell immobilization. (a) The first stage consists of the generation of a microstructured silicon master presenting the negative geometry desired for the PDMS stamps. (b) The second stage is dedicated to the PDMS stamp molding. Liquid PDMS is flowed over the silicon master and reticulated for 1 h at 80 °C. (c) Living cells are finally assembled inside the micropatterns of the PDMS stamp by convective and capillary deposition, forming a cell array.
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using deep reactive ion etching (RIE). The patterns of the silicon master are squares, 4.0 µm 4.5 µm 5.0 µm 5.5 µm 6.0 µm O O 1.5–6 µm wide, with a pitch of 0.5 µm Si Si Si Living cell and a depth ranging from 1 to 4 µm. The c PDMS Convective and measurements of the silicon master should capillary assembly be verified using profilometry. These pattern geometries allow immobilization of a wide range of microorganisms of different sizes (Fig. 1a). For example, patterns with a width of 4–5 µm and a depth of 4 µm are best suited for yeast cells such as C. albicans or S. cerevisiae, does not induce any chemical modification of the cells in contact whereas patterns 1.5–2 µm wide with a depth of 2 µm can be used with it, and it can be used with straight or inverted optical or to image smaller cells such as spores of fungi or round bacteria. fluorescence microscopes that are combined with AFMs. Technical details of the procedure to generate a patterned glass For the second stage of fabricating a PDMS stamp, a PDMS prepolymer solution is cured on the silicon master produced in stage and chromium mask and transfer the patterns to a silicon master, one, and then it is demolded to obtain the microstructured PDMS to fabricate PDMS stamps molded on the silicon master and to stamps (Fig. 1b). The deposition of cells into the microstructured assemble arrays of living cells into the microstructured PDMS PDMS stamps is accomplished using convective and capillary stamps are described below. We demonstrate the type of AFM deposition. Adjustment of parameters such as temperature, humid- results obtained on living cells by showing the results obtained from the medically important yeast pathogen C. albicans, immoity, translation speed and contact angle will ensure a high rate of cell trapping, and they do not affect the cell interface, as cells are bilized into PDMS microstructures. The versatility of this proput back into liquid immediately after the procedure. This stage tocol has been demonstrated for other microorganisms14–17. We anticipate that this approach will be useful for researchers can also be done manually if highly organized arrays of cells are not mandatory for the AFM experiments. The filling rate and the interested in studying microorganisms with AFM at a populaquality of the PDMS stamp can easily be verified by optical micro- tion scale, as well as for biophysicists interested in generating cell scopy. PDMS, being a biocompatible and transparent polymer23,24, arrays for applications in diagnostic or biodetection.
MATERIALS REAGENTS • AZ ECI 3012 photoresist (Microchemicals) ! CAUTION This reagent is a flammable liquid and vapor; it causes serious eye damage and may cause respiratory irritation. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. Avoid breathing vapors or mists. • Hexamethyldisilazane (Sigma-Aldrich) ! CAUTION This reagent is a highly flammable liquid and vapor; it is harmful if inhaled or swallowed, and it is toxic on contact with skin. It also causes severe eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. • Microposit MF CD-26 developer (Shipley) ! CAUTION This reagent is toxic on contact with skin; it causes eye damage and may cause respiratory irritation. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. Avoid breathing vapor or mists. • Octadecyltrichlorosilane in liquid phase (Sigma-Aldrich) ! CAUTION This reagent causes severe skin burns and eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. • Polydimethylsiloxane elastomer Sylgard 184 silicone elastomer (Dow Corning)
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• C. albicans (strain from ABC Platform) or other cells of interest • Yeast peptone dextrose (YPD) broth (Difco) • Sodium acetate (Sigma-Aldrich) • CaCl2 and MnCl2 (Sigma-Aldrich) • Glacial acetic acid (Sigma-Aldrich) ! CAUTION This reagent is a flammable liquid and vapor, and it causes skin burns and eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. EQUIPMENT • CleWin software for mask designing • Mask writing Heidelberg DWL 200 (Heidelberg Instruments) • Oxygen plasma Tepla 300 (PVA TePla America) • Coating/developing machine EVG 120 (EVG Group) • Production mask aligner MA 150 (Suss Microtech) • Inductively coupled plasma-RIE Multiplex Alcatel AMS4200 (Alcatel Micro Machining Systems) • Incubator (Memmert) • Autoclave • Incubator-shaker MAXQ4000 (Fisher Scientific) • Centrifuge Sorvall ST16R (Fisher Scientific)
protocol • Shaker Vortex Top-Mix 1 (Fisher Scientific) • Nanowizard III BioScience atomic force microscope (JPK Instruments) mounted on an inverted optical microscope (Zeiss Axio Observer) equipped with a FireWire CCD color camera (Imaging Source), with 20×, 40× and 50× objectives REAGENT SETUP Cell culture and cell treatment From freshly plated cells, grow C. albicans yeasts cells in YPD broth at 30 °C with agitation (180 rpm) for 18–20 h.
Collect the cells by centrifugation at 4,500g for 3 min at room temperature (20 °C), and rinse them two times in acetate buffer. CRITICAL Cell culture and cell treatment vary according to the type of microorganisms studied. Cell solutions should be freshly prepared before AFM experiments. Sodium acetate buffer For AFM experiments, prepare a sodium acetate buffer solution containing 18 mM sodium acetate, 1 mM CaCl2 and 1 mM MnCl2. Adjust the pH of the solution to 5.2 with glacial acetic acid. The solution can be stored at 4 °C for 2 months.
PROCEDURE Generation of the silicon wafer ● TIMING ~1 week 1| Design the desired micropatterns of the silicon master using the CleWin software (see Supplementary Data for a CleWin file of the design used).
© 2014 Nature America, Inc. All rights reserved.
2| Write the patterns using laser lithography (Heidelberg DWL 200) to make a glass and chromium mask. PAUSE POINT The mask can be stored at room temperature for several months in ambient conditions at 20 °C and 40% humidity (no degradation with time). 3| Clean a virgin silicon wafer under oxygen (pressure of 1.7 mBar) plasma for 15 min at 800 W, using the oxygen plasma Tepla 300. The temperature of the substrate during this step is 20 °C at the beginning of the procedure, and it is 80 °C at the end. 4| Deposit hexamethyldisilazane in solution on the clean silicon master to promote adherence of the photoresist. 5| Deposit the photoresist AZ ECI 3012 on the silicon master using the EVG 120 automatic coating/developing machine (5 s of deposition and 30 s of spinning), and bake it for 60 s at 90 °C on a hot plate (baking is included in the EVG 120 coating recipe). 6| Expose the silicon master covered by the photoresist through the glass and chromium mask using the mask aligner MA 150 for 10 s. CRITICAL STEP During exposition, a crucial parameter is the type of contact between the glass and chromium mask and the silicon master. This parameter is chosen on the mask aligner MA 150. The stronger the contact between the mask and the wafer, the weaker the diffraction of the UV during exposition, and the better the resolution of the patterns. For small patterns (under 5 µm), it is necessary to use the strongest mode of contact between the mask and the wafer, called vacuum contact. In this mode, the wafer is pushed toward the mask, a joint is applied around the wafer and the air between the mask and the wafer is pumped out to generate vacuum. 7| After exposure, bake the silicon master for 60 s at 110 °C on a standard hot plate in ambient air (20 °C and 40% humidity) to complete polymerization of the exposed photoresist. 8| Shape the patterns by dissolving the exposed photoresist in a solution of MF CD-26 developer for 20 s. CRITICAL STEP During development, a crucial parameter is time. Indeed, if the development is too long, the MF CD-26 solution starts to attack the photoresist patterns, leading to the removal of the smallest ones. 9| Rinse both faces of the silicon master using deionized H2O, and dry them under nitrogen. 10| Perform RIE on the silicon master using the Multiplex Alcatel AMS 4200. This step must be realized under plasma of sulfur hexafluoride (SF6, 200 sccm) and octafluorocyclobutane (C4F8, 400 sccm) at a pressure of 0.07 mBar and a power of 2,800 W (inductively coupled plasma). 11| Remove the remaining photoresist from the silicon master under oxygen (pressure of 1.7 mBar) plasma for 15 min at 800 W, using the oxygen plasma Tepla 300. The temperature of the substrate during this step is 20 °C at the beginning of the procedure, and it is 80 °C at the end. 12| Put the silicon master in octadecyltrichlorosilane in liquid phase in order to render the silicon wafer anti-adhesive. PAUSE POINT The silicon master is generated only once, and it can be used hundreds of times. It can be stored in ambient air (20 °C and 40% humidity) for several years when protected in an individual plastic container. nature protocols | VOL.10 NO.1 | 2015 | 201
protocol Fabrication of a microstructured PDMS stamp ● TIMING ~3 h 13| Prepare a PDMS pre-polymer solution containing a mixture in a 10:1 mass ration of PDMS oligomers and a reticular agent (Sylgard kit 184). 14| Degas the prepared solution under vacuum. 15| Deposit the degassed PDMS solution on the silicon master. CRITICAL STEP During this step, bubbles can form, and they may thus cause problems in the microstructures in the PDMS stamp. To avoid this, the silicon master with the PDMS can be degassed again under vacuum. 16| Cure the PDMS solution on the silicon master for 1 h at 80 °C, in ambient air (40% humidity). PAUSE POINT The PDMS cured on the silicon master can be stored at room temperature for several months. To avoid contamination, it is recommended to store the PDMS stamps at room temperature in ambient air, molded on the silicon master.
© 2014 Nature America, Inc. All rights reserved.
17| Cut with a scalpel and demold the microstructured PDMS motif. CRITICAL STEP Cutting a PDMS motif off the silicon master can be crucial; the scalpel must be used gently to avoid scratching or splitting of the silicon master. Assembly of living cells in the microstructured PDMS stamps ● TIMING ~20 min 18| Deposit a PDMS stamp bearing the microstructured motif on a freshly cleaned glass slide. 19| Deposit a 60-µl droplet of the cell suspension on the PDMS stamp. CRITICAL STEP The PDMS stamp is hydrophobic, and the droplet of cells sometimes does not cover up the entire PDMS stamp. To avoid this problem, it is possible to render it hydrophilic by briefly activating it under oxygen plasma for 30 s at 200 W. 20| Assemble the cells into the PDMS microstructure. This can be performed in two different ways: on a convective and capillary setup (option A) or manually (option B). (A) On a convective and capillary setup (i) Drag a droplet of the C. albicans cells onto the PDMS stamp at a given temperature (30 °C), humidity (45%) and translation speed (2 µm/s) using a motorized linear stage. (B) Manually (i) Use a coverslip to drag the droplet of cells several times onto the PDMS stamp. ? TROUBLESHOOTING 21| Use an atomic force microscope with an inverted optical microscope to verify the filling of the wells. If the filling is good, proceed to performing an AFM experiment with the whole PDMS stamp under liquid. Use an AFM procedure adapted to the types of measurements needed on the samples (imaging, probing of nanomechanical or adhesive properties, and multiparametric imaging)18,25. If the wells are not filled, repeat Steps 19–21. ? TROUBLESHOOTING Cells have not filled the PDMS microstructures (Step 20B(i)) Filling of the microstructured wells is a crucial step, whose success depends on the convective and capillary parameters, or on the type of microorganisms used. If after the convective and capillary assembly no cells or very few cells have filled the PDMS microstructured wells, it is possible that the convective and capillary assembly was too fast; hence, reducing the speed can lead to better results. It can also be caused by an insufficient concentration of cells in the droplet. In this case, the cell suspension can be concentrated by centrifugation, and it can be resuspended in a smaller volume of the buffer used, in order to increase the probability of filling the wells. Finally, another possible reason is the surface hydrophobicity of cells. In C. albicans cells, which are very adhesive, working with hydrophobic PDMS stamps is better. However, in other yeast strains, such as S. cerevisiae, that present less adhesins at the surface, a hydrophobic surface will keep them from filling the wells. In this case, we advise working with hydrophilic stamps. Making the stamp hydrophilic can easily be achieved by UV-O3 or O2 plasma treatment. ● TIMING Steps 1–12, generation of the silicon wafer: ~1 week Steps 13–17, fabrication of a microstructured PDMS stamp: ~3 h Steps 18–21, assembly of living cells in the microstructured PDMS stamps: ~20 min 202 | VOL.10 NO.1 | 2015 | nature protocols
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ANTICIPATED RESULTS The immobilization protocol described here (Fig. 1) represents a versatile and nondenaturing method to immobilize living cells of different sizes (Fig. 2), and therefore of different types, without modifying their interfaces. Figure 2 shows PDMS stamps that have been characterized using AFM; cross-sections indicate the different sizes that can be obtained on the PDMS stamp. A key advantage of this protocol is that it allows the generation of cell arrays, which enables an increased sample size and hence statistical power, and gives access to population heterogeneity data. Figure 3 shows the results obtained using this protocol to generate a cell array of C. albicans, with AFM imaging using Quantitative Imaging mode from JPK Instruments14,26. These data show that this protocol generates cell arrays, as all the 16 wells of 4.5 × 4.5 µm2 (830 pixels per well) were filled with cells. It also shows the heterogeneity that exists between cells. Indeed, as we can see in this figure, some cells are higher than others, present a bigger diameter, or are in a budding process. Force measurements can be performed on each of the cells present in this 2 µm array, thus giving access to their mechanical or adhesive properties. By using multiparametric imaging (Quantitative Imaging mode, in this case), one image of an entire cell | Figure 3 Imaging of a C. albicans cell array. AFM 3D height image (scan array can lead to the quantification of the nanomechanical size = 40 µm, z-range = 2 µm) of an array of 4 × 4 microstructured PDMS properties (i.e., Young modulus) and the adhesive properties wells, exhibiting a filling rate of 100%, in sodium acetate buffer. 45 0
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protocol of several cells at the same time. Figure 4 shows the result a b c 2 µm 4 MPa 1 nN of such an experiment. Figure 4a shows a height image of a cell array of 10 × 10 wells of 5 × 5 µm2 (165 pixels per well), presenting a filling rate of 85%. Figure 4b shows the elasticity map corresponding to the height image, and Figure 3c presents the adhesion image corresponding to 20 µm 20 µm 20 µm 0 0 0 the height image. This set of data clearly shows the power Figure 4 | Multiparametric imaging of a C. albicans array. (a) AFM 2D height of such a protocol used in combination with advanced AFM image (scan size = 100 µm) of an array of 10 × 10 microstructured PDMS modes such as multiparametric imaging. Indeed, in one wells, exhibiting a filling rate of 85%. (b,c) Elasticity map (b) and adhesion acquisition performed (165 pixels per well, 2.64 s of map (c) corresponding to the height image presented in a. acquisition per wells), 85 cells were imaged, and they were probed for their nanomechanical properties and for their surface adhesive properties. It is clearly visible on Figure 4b,c that the same population of cells exhibits heterogeneity; this point was first evoked in our study in 2011 (ref. 11). Thus, cells of C. albicans that have grown in the same culture medium present different surface adhesive properties and cell wall Young modulus. If needed, high-magnification and high-resolution multiparametric data (height, adhesion and stiffness) can be recorded on single cells27. Owing to the versatility of this protocol, similar results can be obtained for microorganisms of different sizes, such as cocci bacteria, other yeast species or algae. This protocol opens an avenue to further developments that could lead to great advances in the field of microbiology. Indeed, the PDMS stamp could be coupled with a microfluidic system to add different substances (such as antifungals) to each well. By probing the nanoscale characteristics of the cells present in these two wells at the same time, a direct comparison of the effects of the two different substances could be performed.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments 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. AUTHOR 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. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Binnig, G., Quate, C.F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–934 (1986). 2. Gerber, C. & Lang, H.P. How the doors to the nanoworld were opened. Nat. Nanotechnol. 1, 3–5 (2006). 3. Louise Meyer, R. et al. Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy 110, 1349–1357 (2010). 4. Doktycz, M.J. et al. AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces. Ultramicroscopy 97, 209–216 (2003). 5. Kasas, S. & Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995). 6. Kailas, L. et al. Immobilizing live bacteria for AFM imaging of cellular processes. Ultramicroscopy 109, 775–780 (2009). 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). 8. Alsteens, D. et al. Structure, cell wall elasticity and polysaccharide properties of living yeasts cells, as probed by AFM. Nanotechnology 19, 384005 (2008). 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).
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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). 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). 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). 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). 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). 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). 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). 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). 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). 19. Dufrêne, Y.F. Atomic force microscopy and chemical force microscopy of microbial cells. Nat. Protoc. 3, 1132–1138 (2008). 20. Francius, G. et al. Stretching polysaccharides on live cells using single-molecule force spectroscopy. Nat. Protoc. 4, 939–946 (2009). 21. Beaussart, A. et al. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 9, 1049–1055 (2014). 22. Roduit, C. et al. OpenFovea: open-source AFM data processing software. Nat. Methods 9, 774–775 (2012). 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). 24. Chabinyc, M.L. et al. An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications. Anal. Chem. 73, 4491–4498 (2001). 25. Liu, S. & Wang, Y. Application of AFM in microbiology: a review. Scanning 32, 61–73 (2010). 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. doi:10.1016/j.nano.2014.07.008 (4 August 2014).
Generation of living cell arrays for atomic force microscopy studies Cécile Formosa1–4, Flavien Pillet2,5, Marion Schiavone1,2,6, Raphaël E Duval3,4,7, Laurence Ressier2,8 & Etienne Dague1,2 1Centre Nationale de la Recherche Scientifique (CNRS), Laboratoire d’Analyse et d’Architecture des Systèmes (LAAS), Toulouse, France. 2Université de Toulouse;
LAAS, Institut des Technologies Avancées en Sciences du Vivant (ITAV), Institute de Pharmacologie et de Biologie Structurale (IPBS), Toulouse, France. 3CNRS, Unité Mixte de Recherche (UMR) 7565, Laboratoire Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Vandœuvre-lès-Nancy, France. 4Université de Lorraine, UMR 7565, Faculté de Pharmacie, Nancy, France. 5CNRS, IPBS, UMR 5089, Toulouse, France. 6L’Institut National de la Recherche Agronomie (INRA), UMR 972 Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Toulouse, France. 7ABC Platform, Nancy, France. 8Université 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. Correspondence should be addressed to E.D. ([email protected]).
© 2014 Nature America, Inc. All rights reserved.
Published online 31 December 2014; doi:10.1038/nprot.2015.004
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.
INTRODUCTION Since its first development in 1986 (ref. 1), AFM has developed into a powerful tool that has opened the doors to the nanoworld2. AFM is particularly well suited for biology, as it allows multiple characterizations (topography, mechanical and adhesive properties) of living cells in their physiological environments. However, a prerequisite for such AFM experiments is the immobilization of the biological samples probed. However, this crucial step is often a challenge, as samples have to be immobilized individually and firmly enough to withstand the lateral forces exerted by the AFM tip, but without altering their cellular integrity. Immobilization of cells for AFM experiments Several techniques have been used to immobilize living cells. Living cells such as microorganisms can be chemically fixed on a solid substrate using glutaraldehyde or (3-aminopropyl)triethox ysilane (APTES)3, or they can be immobilized on gelatin-coated surfaces4. However, these techniques can, respectively, modify the interface of the biological sample, or they can pollute the AFM tips, leading to artifacts. Another strategy is to trap roundshaped cells such as bacterial cocci and yeast cells in the pores of polycarbonate membranes by filtration5 or in lithographically patterned substrates by gentle drying6. The filtration technique has been widely used over recent years7–10, although it is timeconsuming and cells can be exposed to mechanical forces when trapped in the pores. We therefore developed a new and versatile strategy in 2011 (refs. 11–13), which consists of immobilizing round single living cells in microstructured PDMS stamps by convective/capillary deposition. We have demonstrated that this generic protocol can be used to immobilize different types of round cells, ranging from small cocci bacteria to yeasts cells and even algae, by tuning the geometry of the PDMS stamp patterns (Supplementary Data).
This approach was also exploited to immobilize yeast cells of different species, Saccharomyces cerevisiae and Candida albicans14–16, as well as spores of Aspergillus fumigatus17. Statistical significance of results obtained from AFM: the new challenge Statistical analysis of results is desirable. As AFM is a tool adapted for single-cell analysis, this technology requires the analysis of multiple cells in order to achieve statistical confidence. This cannot be performed using techniques such as immobilization in a pore filter, as the deposition of the cells on the surface is random and the rate of filled pores is low and not controlled. By using our method of immobilization, it is possible to generate arrays of cells; therefore, AFM results on an array of 100 cells can be performed using different AFM modes, such as multiparametric imaging, chemical force microscopy, single-molecule force spectroscopy and single-cell force spectroscopy14,18–21. Such setups are effectively ‘lab chips’ for AFM analysis. With the progress made in AFM data processing software22, it is thus possible to take all the data acquired to analyze in a reasonable time period, so as to generate relevant and statistically significant results for biological studies. Overview of the procedure Immobilization of living cells in PDMS stamps involves three sequential stages. The first stage is the generation of a glass and chromium mask that harbors microstructured patterns, and the transfer of these patterns onto a silicon wafer. The second stage consists of the preparation of a corresponding PDMS stamp. Finally, the third stage is the assembly of the living cells into the PDMS microstructured stamps. The generation of a silicon master is achieved by photolithography, followed by pattern transfer nature protocols | VOL.10 NO.1 | 2015 | 199
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Figure 1 | Schematics of the protocol for living cell immobilization. (a) The first stage consists of the generation of a microstructured silicon master presenting the negative geometry desired for the PDMS stamps. (b) The second stage is dedicated to the PDMS stamp molding. Liquid PDMS is flowed over the silicon master and reticulated for 1 h at 80 °C. (c) Living cells are finally assembled inside the micropatterns of the PDMS stamp by convective and capillary deposition, forming a cell array.
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using deep reactive ion etching (RIE). The patterns of the silicon master are squares, 4.0 µm 4.5 µm 5.0 µm 5.5 µm 6.0 µm O O 1.5–6 µm wide, with a pitch of 0.5 µm Si Si Si Living cell and a depth ranging from 1 to 4 µm. The c PDMS Convective and measurements of the silicon master should capillary assembly be verified using profilometry. These pattern geometries allow immobilization of a wide range of microorganisms of different sizes (Fig. 1a). For example, patterns with a width of 4–5 µm and a depth of 4 µm are best suited for yeast cells such as C. albicans or S. cerevisiae, does not induce any chemical modification of the cells in contact whereas patterns 1.5–2 µm wide with a depth of 2 µm can be used with it, and it can be used with straight or inverted optical or to image smaller cells such as spores of fungi or round bacteria. fluorescence microscopes that are combined with AFMs. Technical details of the procedure to generate a patterned glass For the second stage of fabricating a PDMS stamp, a PDMS prepolymer solution is cured on the silicon master produced in stage and chromium mask and transfer the patterns to a silicon master, one, and then it is demolded to obtain the microstructured PDMS to fabricate PDMS stamps molded on the silicon master and to stamps (Fig. 1b). The deposition of cells into the microstructured assemble arrays of living cells into the microstructured PDMS PDMS stamps is accomplished using convective and capillary stamps are described below. We demonstrate the type of AFM deposition. Adjustment of parameters such as temperature, humid- results obtained on living cells by showing the results obtained from the medically important yeast pathogen C. albicans, immoity, translation speed and contact angle will ensure a high rate of cell trapping, and they do not affect the cell interface, as cells are bilized into PDMS microstructures. The versatility of this proput back into liquid immediately after the procedure. This stage tocol has been demonstrated for other microorganisms14–17. We anticipate that this approach will be useful for researchers can also be done manually if highly organized arrays of cells are not mandatory for the AFM experiments. The filling rate and the interested in studying microorganisms with AFM at a populaquality of the PDMS stamp can easily be verified by optical micro- tion scale, as well as for biophysicists interested in generating cell scopy. PDMS, being a biocompatible and transparent polymer23,24, arrays for applications in diagnostic or biodetection.
MATERIALS REAGENTS • AZ ECI 3012 photoresist (Microchemicals) ! CAUTION This reagent is a flammable liquid and vapor; it causes serious eye damage and may cause respiratory irritation. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. Avoid breathing vapors or mists. • Hexamethyldisilazane (Sigma-Aldrich) ! CAUTION This reagent is a highly flammable liquid and vapor; it is harmful if inhaled or swallowed, and it is toxic on contact with skin. It also causes severe eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. • Microposit MF CD-26 developer (Shipley) ! CAUTION This reagent is toxic on contact with skin; it causes eye damage and may cause respiratory irritation. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. Avoid breathing vapor or mists. • Octadecyltrichlorosilane in liquid phase (Sigma-Aldrich) ! CAUTION This reagent causes severe skin burns and eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. • Polydimethylsiloxane elastomer Sylgard 184 silicone elastomer (Dow Corning)
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• C. albicans (strain from ABC Platform) or other cells of interest • Yeast peptone dextrose (YPD) broth (Difco) • Sodium acetate (Sigma-Aldrich) • CaCl2 and MnCl2 (Sigma-Aldrich) • Glacial acetic acid (Sigma-Aldrich) ! CAUTION This reagent is a flammable liquid and vapor, and it causes skin burns and eye damage. When handling this reagent, wear protective gloves, protective clothing, eye protection and face protection. EQUIPMENT • CleWin software for mask designing • Mask writing Heidelberg DWL 200 (Heidelberg Instruments) • Oxygen plasma Tepla 300 (PVA TePla America) • Coating/developing machine EVG 120 (EVG Group) • Production mask aligner MA 150 (Suss Microtech) • Inductively coupled plasma-RIE Multiplex Alcatel AMS4200 (Alcatel Micro Machining Systems) • Incubator (Memmert) • Autoclave • Incubator-shaker MAXQ4000 (Fisher Scientific) • Centrifuge Sorvall ST16R (Fisher Scientific)
protocol • Shaker Vortex Top-Mix 1 (Fisher Scientific) • Nanowizard III BioScience atomic force microscope (JPK Instruments) mounted on an inverted optical microscope (Zeiss Axio Observer) equipped with a FireWire CCD color camera (Imaging Source), with 20×, 40× and 50× objectives REAGENT SETUP Cell culture and cell treatment From freshly plated cells, grow C. albicans yeasts cells in YPD broth at 30 °C with agitation (180 rpm) for 18–20 h.
Collect the cells by centrifugation at 4,500g for 3 min at room temperature (20 °C), and rinse them two times in acetate buffer. CRITICAL Cell culture and cell treatment vary according to the type of microorganisms studied. Cell solutions should be freshly prepared before AFM experiments. Sodium acetate buffer For AFM experiments, prepare a sodium acetate buffer solution containing 18 mM sodium acetate, 1 mM CaCl2 and 1 mM MnCl2. Adjust the pH of the solution to 5.2 with glacial acetic acid. The solution can be stored at 4 °C for 2 months.
PROCEDURE Generation of the silicon wafer ● TIMING ~1 week 1| Design the desired micropatterns of the silicon master using the CleWin software (see Supplementary Data for a CleWin file of the design used).
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2| Write the patterns using laser lithography (Heidelberg DWL 200) to make a glass and chromium mask. PAUSE POINT The mask can be stored at room temperature for several months in ambient conditions at 20 °C and 40% humidity (no degradation with time). 3| Clean a virgin silicon wafer under oxygen (pressure of 1.7 mBar) plasma for 15 min at 800 W, using the oxygen plasma Tepla 300. The temperature of the substrate during this step is 20 °C at the beginning of the procedure, and it is 80 °C at the end. 4| Deposit hexamethyldisilazane in solution on the clean silicon master to promote adherence of the photoresist. 5| Deposit the photoresist AZ ECI 3012 on the silicon master using the EVG 120 automatic coating/developing machine (5 s of deposition and 30 s of spinning), and bake it for 60 s at 90 °C on a hot plate (baking is included in the EVG 120 coating recipe). 6| Expose the silicon master covered by the photoresist through the glass and chromium mask using the mask aligner MA 150 for 10 s. CRITICAL STEP During exposition, a crucial parameter is the type of contact between the glass and chromium mask and the silicon master. This parameter is chosen on the mask aligner MA 150. The stronger the contact between the mask and the wafer, the weaker the diffraction of the UV during exposition, and the better the resolution of the patterns. For small patterns (under 5 µm), it is necessary to use the strongest mode of contact between the mask and the wafer, called vacuum contact. In this mode, the wafer is pushed toward the mask, a joint is applied around the wafer and the air between the mask and the wafer is pumped out to generate vacuum. 7| After exposure, bake the silicon master for 60 s at 110 °C on a standard hot plate in ambient air (20 °C and 40% humidity) to complete polymerization of the exposed photoresist. 8| Shape the patterns by dissolving the exposed photoresist in a solution of MF CD-26 developer for 20 s. CRITICAL STEP During development, a crucial parameter is time. Indeed, if the development is too long, the MF CD-26 solution starts to attack the photoresist patterns, leading to the removal of the smallest ones. 9| Rinse both faces of the silicon master using deionized H2O, and dry them under nitrogen. 10| Perform RIE on the silicon master using the Multiplex Alcatel AMS 4200. This step must be realized under plasma of sulfur hexafluoride (SF6, 200 sccm) and octafluorocyclobutane (C4F8, 400 sccm) at a pressure of 0.07 mBar and a power of 2,800 W (inductively coupled plasma). 11| Remove the remaining photoresist from the silicon master under oxygen (pressure of 1.7 mBar) plasma for 15 min at 800 W, using the oxygen plasma Tepla 300. The temperature of the substrate during this step is 20 °C at the beginning of the procedure, and it is 80 °C at the end. 12| Put the silicon master in octadecyltrichlorosilane in liquid phase in order to render the silicon wafer anti-adhesive. PAUSE POINT The silicon master is generated only once, and it can be used hundreds of times. It can be stored in ambient air (20 °C and 40% humidity) for several years when protected in an individual plastic container. nature protocols | VOL.10 NO.1 | 2015 | 201
protocol Fabrication of a microstructured PDMS stamp ● TIMING ~3 h 13| Prepare a PDMS pre-polymer solution containing a mixture in a 10:1 mass ration of PDMS oligomers and a reticular agent (Sylgard kit 184). 14| Degas the prepared solution under vacuum. 15| Deposit the degassed PDMS solution on the silicon master. CRITICAL STEP During this step, bubbles can form, and they may thus cause problems in the microstructures in the PDMS stamp. To avoid this, the silicon master with the PDMS can be degassed again under vacuum. 16| Cure the PDMS solution on the silicon master for 1 h at 80 °C, in ambient air (40% humidity). PAUSE POINT The PDMS cured on the silicon master can be stored at room temperature for several months. To avoid contamination, it is recommended to store the PDMS stamps at room temperature in ambient air, molded on the silicon master.
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17| Cut with a scalpel and demold the microstructured PDMS motif. CRITICAL STEP Cutting a PDMS motif off the silicon master can be crucial; the scalpel must be used gently to avoid scratching or splitting of the silicon master. Assembly of living cells in the microstructured PDMS stamps ● TIMING ~20 min 18| Deposit a PDMS stamp bearing the microstructured motif on a freshly cleaned glass slide. 19| Deposit a 60-µl droplet of the cell suspension on the PDMS stamp. CRITICAL STEP The PDMS stamp is hydrophobic, and the droplet of cells sometimes does not cover up the entire PDMS stamp. To avoid this problem, it is possible to render it hydrophilic by briefly activating it under oxygen plasma for 30 s at 200 W. 20| Assemble the cells into the PDMS microstructure. This can be performed in two different ways: on a convective and capillary setup (option A) or manually (option B). (A) On a convective and capillary setup (i) Drag a droplet of the C. albicans cells onto the PDMS stamp at a given temperature (30 °C), humidity (45%) and translation speed (2 µm/s) using a motorized linear stage. (B) Manually (i) Use a coverslip to drag the droplet of cells several times onto the PDMS stamp. ? TROUBLESHOOTING 21| Use an atomic force microscope with an inverted optical microscope to verify the filling of the wells. If the filling is good, proceed to performing an AFM experiment with the whole PDMS stamp under liquid. Use an AFM procedure adapted to the types of measurements needed on the samples (imaging, probing of nanomechanical or adhesive properties, and multiparametric imaging)18,25. If the wells are not filled, repeat Steps 19–21. ? TROUBLESHOOTING Cells have not filled the PDMS microstructures (Step 20B(i)) Filling of the microstructured wells is a crucial step, whose success depends on the convective and capillary parameters, or on the type of microorganisms used. If after the convective and capillary assembly no cells or very few cells have filled the PDMS microstructured wells, it is possible that the convective and capillary assembly was too fast; hence, reducing the speed can lead to better results. It can also be caused by an insufficient concentration of cells in the droplet. In this case, the cell suspension can be concentrated by centrifugation, and it can be resuspended in a smaller volume of the buffer used, in order to increase the probability of filling the wells. Finally, another possible reason is the surface hydrophobicity of cells. In C. albicans cells, which are very adhesive, working with hydrophobic PDMS stamps is better. However, in other yeast strains, such as S. cerevisiae, that present less adhesins at the surface, a hydrophobic surface will keep them from filling the wells. In this case, we advise working with hydrophilic stamps. Making the stamp hydrophilic can easily be achieved by UV-O3 or O2 plasma treatment. ● TIMING Steps 1–12, generation of the silicon wafer: ~1 week Steps 13–17, fabrication of a microstructured PDMS stamp: ~3 h Steps 18–21, assembly of living cells in the microstructured PDMS stamps: ~20 min 202 | VOL.10 NO.1 | 2015 | nature protocols
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ANTICIPATED RESULTS The immobilization protocol described here (Fig. 1) represents a versatile and nondenaturing method to immobilize living cells of different sizes (Fig. 2), and therefore of different types, without modifying their interfaces. Figure 2 shows PDMS stamps that have been characterized using AFM; cross-sections indicate the different sizes that can be obtained on the PDMS stamp. A key advantage of this protocol is that it allows the generation of cell arrays, which enables an increased sample size and hence statistical power, and gives access to population heterogeneity data. Figure 3 shows the results obtained using this protocol to generate a cell array of C. albicans, with AFM imaging using Quantitative Imaging mode from JPK Instruments14,26. These data show that this protocol generates cell arrays, as all the 16 wells of 4.5 × 4.5 µm2 (830 pixels per well) were filled with cells. It also shows the heterogeneity that exists between cells. Indeed, as we can see in this figure, some cells are higher than others, present a bigger diameter, or are in a budding process. Force measurements can be performed on each of the cells present in this 2 µm array, thus giving access to their mechanical or adhesive properties. By using multiparametric imaging (Quantitative Imaging mode, in this case), one image of an entire cell | Figure 3 Imaging of a C. albicans cell array. AFM 3D height image (scan array can lead to the quantification of the nanomechanical size = 40 µm, z-range = 2 µm) of an array of 4 × 4 microstructured PDMS properties (i.e., Young modulus) and the adhesive properties wells, exhibiting a filling rate of 100%, in sodium acetate buffer. 45 0
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protocol of several cells at the same time. Figure 4 shows the result a b c 2 µm 4 MPa 1 nN of such an experiment. Figure 4a shows a height image of a cell array of 10 × 10 wells of 5 × 5 µm2 (165 pixels per well), presenting a filling rate of 85%. Figure 4b shows the elasticity map corresponding to the height image, and Figure 3c presents the adhesion image corresponding to 20 µm 20 µm 20 µm 0 0 0 the height image. This set of data clearly shows the power Figure 4 | Multiparametric imaging of a C. albicans array. (a) AFM 2D height of such a protocol used in combination with advanced AFM image (scan size = 100 µm) of an array of 10 × 10 microstructured PDMS modes such as multiparametric imaging. Indeed, in one wells, exhibiting a filling rate of 85%. (b,c) Elasticity map (b) and adhesion acquisition performed (165 pixels per well, 2.64 s of map (c) corresponding to the height image presented in a. acquisition per wells), 85 cells were imaged, and they were probed for their nanomechanical properties and for their surface adhesive properties. It is clearly visible on Figure 4b,c that the same population of cells exhibits heterogeneity; this point was first evoked in our study in 2011 (ref. 11). Thus, cells of C. albicans that have grown in the same culture medium present different surface adhesive properties and cell wall Young modulus. If needed, high-magnification and high-resolution multiparametric data (height, adhesion and stiffness) can be recorded on single cells27. Owing to the versatility of this protocol, similar results can be obtained for microorganisms of different sizes, such as cocci bacteria, other yeast species or algae. This protocol opens an avenue to further developments that could lead to great advances in the field of microbiology. Indeed, the PDMS stamp could be coupled with a microfluidic system to add different substances (such as antifungals) to each well. By probing the nanoscale characteristics of the cells present in these two wells at the same time, a direct comparison of the effects of the two different substances could be performed.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments 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. AUTHOR 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. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Binnig, G., Quate, C.F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–934 (1986). 2. Gerber, C. & Lang, H.P. How the doors to the nanoworld were opened. Nat. Nanotechnol. 1, 3–5 (2006). 3. Louise Meyer, R. et al. Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy 110, 1349–1357 (2010). 4. Doktycz, M.J. et al. AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces. Ultramicroscopy 97, 209–216 (2003). 5. Kasas, S. & Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995). 6. Kailas, L. et al. Immobilizing live bacteria for AFM imaging of cellular processes. Ultramicroscopy 109, 775–780 (2009). 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). 8. Alsteens, D. et al. Structure, cell wall elasticity and polysaccharide properties of living yeasts cells, as probed by AFM. Nanotechnology 19, 384005 (2008). 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).
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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). 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). 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). 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). 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). 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). 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). 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). 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). 19. Dufrêne, Y.F. Atomic force microscopy and chemical force microscopy of microbial cells. Nat. Protoc. 3, 1132–1138 (2008). 20. Francius, G. et al. Stretching polysaccharides on live cells using single-molecule force spectroscopy. Nat. Protoc. 4, 939–946 (2009). 21. Beaussart, A. et al. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 9, 1049–1055 (2014). 22. Roduit, C. et al. OpenFovea: open-source AFM data processing software. Nat. Methods 9, 774–775 (2012). 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). 24. Chabinyc, M.L. et al. An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications. Anal. Chem. 73, 4491–4498 (2001). 25. Liu, S. & Wang, Y. Application of AFM in microbiology: a review. Scanning 32, 61–73 (2010). 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. doi:10.1016/j.nano.2014.07.008 (4 August 2014).
