Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Ligand functionalization of titanium nanopattern enables the analysis of cell–ligand interactions by super-resolution microscopy

Nature Protocols volume 17, pages 2275–2306 (2022)Cite this article

Subjects

Abstract

The spatiotemporal aspects of early signaling events during interactions between cells and their environment dictate multiple downstream outcomes. While advances in nanopatterning techniques have allowed the isolation of these signaling events, a major limitation of conventional nanopatterning methods is its dependence on gold (Au) or related materials that plasmonically quench fluorescence and, thus, are incompatible with super-resolution fluorescence microscopy. Here we describe a novel method that integrates nanopatterning with single-molecule resolution fluorescence imaging, thus enabling mechanistic dissection of molecular-scale signaling events in conjunction with nanoscale geometry manipulation. Our method exploits nanofabricated titanium (Ti) whose oxide (TiO2) is a dielectric material with no plasmonic effects. We describe the surface chemistry for decorating specific ligands such as cyclo-RGD (arginine, glycine and aspartate: a ligand for fibronectin-binding integrins) on TiO2 nanoline and nanodot substrates, and demonstrate the ability to perform dual-color super-resolution imaging on these patterns. Ti nanofabrication is similar to other metallic materials like Au, while the functionalization of TiO2 is relatively fast, safe, economical, easy to set up with commonly available reagents, and robust against environmental parameters such as humidity. Fabrication of nanopatterns takes ~2–3 d, preparation for functionalization ~1.5–2 d, and functionalization 3 h, after which cell culture and imaging experiments can be performed. We suggest that this method may facilitate the interrogation of nanoscale geometry and force at single-molecule resolution, and should find ready applications in early detection and interpretation of physiochemical signaling events at the cell membrane in the fields of cell biology, immunology, regenerative medicine, and related fields.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Nanofabrication of Ti nanopatterns on glass substrates.
Fig. 2: Functionalization of Ti substrates with cyclo-RGD and passivation of surrounding glass with supported lipid bilayers (SLBs).
Fig. 3: Nanofabrication inspection examples.
Fig. 4: FRAP to test fluidic nature of SLBs formed.
Fig. 5: Lipid thin-film deposition in round bottom flask.
Fig. 6: Ultrasonication steps for obtaining SUV solution.
Fig. 7: Detailed timeline for nanopattern fabrication, functionalization, cell seeding and super-resolution imaging.
Fig. 8: Charaterization of nanopatterns by AFM.
Fig. 9: Functionalized substrates visualized using confocal and dSTORM microscopy.
Fig. 10: Reconstructed PALM and dSTORM super-resolution images.

Similar content being viewed by others

Data availability

No original code has been used for this paper. The raw data that support the anticipated results are available at figshare: https://doi.org/10.6084/m9.figshare.19337648. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Geiger, B., Spatz, J. P. & Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking cell–matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Doyle, A. D. & Yamada, K. M. Mechanosensing via cell-matrix adhesions in 3D microenvironments. Exp. Cell Res. 343, 60–66 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Changede, R., Cai, H., Wind, S. J. & Sheetz, M. P. Integrin nanoclusters can bridge thin matrix fibres to form cell–matrix adhesions. Nat. Mater. 18, 1366–1375 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Erickson, B. et al. Nanoscale structure of type I collagen fibrils: quantitative measurement of D-spacing. Biotechnol. J. 8, 117–126 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Früh, S. M., Schoen, I., Ries, J. & Vogel, V. Molecular architecture of native fibronectin fibrils. Nat. Commun. 6, 1–10 (2015).

    Article  Google Scholar 

  7. Mossman, K. D., Campi, G., Groves, J. T. & Dustin, M. L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Lee, K.-H. et al. T cell receptor signaling precedes immunological synapse formation. Science 295, 1539–1542 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Morrison, S. J. & Kimble, J. Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 441, 1068–1074 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Moya, I. M. & Halder, G. Hippo–YAP/TAZ signalling in organ regeneration and regenerative medicine. Nat. Rev. Mol. Cell Biol. 20, 211–226 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Papenfort, K. & Bassler, B. L. Quorum sensing signal–response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14, 576–588 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lindner, M. et al. A fast and simple contact printing approach to generate 2D protein nanopatterns. Front. Chem. 7, 655 (2019).

    Article  Google Scholar 

  14. Huang, Y., Tran, H. & Ober, C. K. High-resolution nanopatterning of free-standing, self-supported helical polypeptide rod brushes via electron beam lithography. ACS Macro Lett. 10, 755–759 (2021).

    Article  PubMed  Google Scholar 

  15. Han, P. et al. Five piconewtons: the difference between osteogenic and adipogenic fate choice in human mesenchymal stem cells. ACS Nano 13, 11129–11143 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Killops, K. L. et al. Nanopatterning biomolecules by block copolymer self-assembly. ACS Macro Lett. 1, 758–763 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. M, A. et al. Activation of integrin function by nanopatterned adhesive interfaces. Chemphyschem 5, 383–388 (2004).

    Article  Google Scholar 

  18. Cai, H. et al. Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering. Nat. Nanotechnol. 13, 610–617 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lohm€, T. et al. Supported membranes embedded with fixed arrays of gold nanoparticles. Nano Lett. 11, 37 (2011).

    Google Scholar 

  20. Cai, H. et al. Molecular occupancy of nanodot arrays. ACS Nano 10, 4173–4183 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chighizola F, M. et al. The glycocalyx affects force loading-dependent mechanotransductive topography sensing at the nanoscale. Preprint at https://www.biorxiv.org/content/10.1101/2021.03.02.433591v2 (2021).

  22. Schulte, C. et al. Conversion of nanoscale topographical information of cluster-assembled zirconia surfaces into mechanotransductive events promotes neuronal differentiation. J. Nanobiotechnol. 14, 1–24 (2016).

    Article  Google Scholar 

  23. Elias, C. N., Lima, J. H. C., Valiev, R. & Meyers, M. A. Biomedical applications of titanium and its alloys. JOM 60, 46–49 (2008). 2008 603.

    Article  CAS  Google Scholar 

  24. Paschalis, E. I. et al. In vitro and in vivo assessment of titanium surface modification for coloring the backplate of the Boston keratoprosthesis. Invest. Ophthalmol. Vis. Sci. 54, 3863–3873 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Sidambe, A. T. & Oh, J. K. Biocompatibility of advanced manufactured titanium implants—a review. Materials 7, 8168–8188 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Quinn, R. K. & Armstrong, N. R. Electrochemical and surface analytical characterization of titanium and titanium hydride thin film electrode oxidation. J. Electrochem. Soc. 125, 1790–1796 (1978).

    Article  CAS  Google Scholar 

  27. Ponader, S. et al. Effects of topographical surface modifications of electron beam melted Ti-6Al-4V titanium on human fetal osteoblasts. J. Biomed. Mater. Res. A 84, 1111–1119 (2008).

    Article  PubMed  Google Scholar 

  28. Haslauer, C. M. et al. In vitro biocompatibility of titanium alloy discs made using direct metal fabrication. Med. Eng. Phys. 32, 645–652 (2010).

    Article  PubMed  Google Scholar 

  29. Guasch, J. et al. Synthesis of binary nanopatterns on hydrogels for initiating cellular responses. Chem. Mater. 28, 1806–1815 (2016).

    Article  CAS  Google Scholar 

  30. Ziental, D. et al. Titanium dioxide nanoparticles: prospects and applications in medicine. Nanomaterials 10, 387 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  31. Rajh, T., Dimitrijevic, N. M. & Rozhkova, E. A. Titanium dioxide nanoparticles in advanced imaging and nanotherapeutics. Methods Mol. Biol. 726, 63–75 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Rechenmacher, F. et al. A molecular toolkit for the functionalization of titanium-based biomaterials that selectively control integrin-mediated cell adhesion. Chemistry 19, 9218–9223 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Svensson, F. G., Daniel, G., Tai, C.-W., Seisenbaeva, G. A. & Kessler, V. G. Titanium phosphonate oxo-alkoxide ‘clusters’: solution stability and facile hydrolytic transformation into nano titania. RSC Adv. 10, 6873–6883 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Eriksson, A. I. K., Edwards, K., Hagfeldt, A. & Hernández, V. A. Physicochemical characterization of phosphopeptide/titanium dioxide interactions employing the quartz crystal microbalance technique. J. Phys. Chem. B 117, 2019–2025 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. Glass, R., Möller, M. & Spatz, J. P. Block copolymer micelle nanolithography. Nanotechnology 14, 1153 (2003).

    Article  CAS  Google Scholar 

  36. Harris, J. M. Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications (Springer Science & Business Media, 1992).

  37. Jain, A., Liu, R., Xiang, Y. K. & Ha, T. Single-molecule pull-down for studying protein interactions. Nat. Protoc. 7, 445–452 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Blümmel, J. et al. Protein repellent properties of covalently attached PEG coatings on nanostructured SiO2-based interfaces. Biomaterials 28, 4739–4747 (2007).

    Article  PubMed  Google Scholar 

  39. Zhu, B., Eurell, T., Gunawan, R. & Leckband, D. Chain-length dependence of the protein and cell resistance of oligo(ethylene glycol)-terminated self-assembled monolayers on gold. J. Biomed. Mater. Res. 56, 406–416 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Cai, H. & Wind, S. J. Improved glass surface passivation for single-molecule nanoarrays. Langmuir 32, 10034–10041 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Persson, F. et al. Lipid-based passivation in nanofluidics. Nano Lett. 12, 2260–2265 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Durisic, N., Laparra-Cuervo, L., Sandoval-Álvarez, Á., Borbely, J. S. & Lakadamyali, M. Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat. Methods 11, 156–162 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. Yuwono, A. H. et al. Diblock copolymer templated nanohybrid thin films of highly ordered TiO2 nanoparticle arrays in PMMA matrix. Chem. Mater. 18, 5876–5889 (2006).

    Article  CAS  Google Scholar 

  45. Polleux, J. et al. Benzyl alcohol and block copolymer micellar lithography: a versatile route to assembling gold and in situ generated titania nanoparticles into uniform binary nanoarrays. ACS Nano 5, 6355–6364 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Whelan, D. R. & Bell, T. D. M. Super-resolution single-molecule localization microscopy: tricks of the trade. J. Phys. Chem. Lett. 6, 374–382 (2015).

    Article  CAS  PubMed  Google Scholar 

  47. Khater, I. M., Nabi, I. R. & Hamarneh, G. A review of super-resolution single-molecule localization microscopy cluster analysis and quantification methods. Patterns 1, 100038 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ovesný, M., Křížek, P., Borkovec, J., Švindrych, Z. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 2389–2390 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Changede, R., Xu, X., Margadant, F. & Sheetz, M. P. Nascent integrin adhesions form on all matrix rigidities after integrin activation. Dev. Cell 35, 614–621 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Wolter, S. et al. rapidSTORM: accurate, fast open-source software for localization microscopy. Nat. Methods 9, 1040–1041 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Nehme, E., Weiss, L. E., Michaeli, T. & Shechtman, Y. Deep-STORM: super-resolution single-molecule microscopy by deep learning. Optica 5, 458–464 (2018).

    Article  CAS  Google Scholar 

  53. Vogel, V. & Sheetz, M. P. Mechanical forces matter in health and disease: from cancer to tissue engineering. Nanotechnology 5, 233–303 (2010).

    Google Scholar 

  54. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  PubMed  Google Scholar 

  55. Zhan, T., Rindtorff, N. & Boutros, M. Wnt signaling in cancer. Oncogene 36, 1461–1473 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Sanchez-Vega, F. et al. Oncogenic signaling pathways in The Cancer Genome Atlas. Cell 173, 321–337.e10 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Pan, L. et al. Higher-order clustering of the transmembrane anchor of DR5 drives signaling. Cell 176, 1477–1489.e14 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. L Berger, R. M. et al. Nanoscale FasL organization on DNA origami to decipher apoptosis signal activation in cells. Small 17, 2101678 (2021).

    Article  Google Scholar 

  59. Aggarwal, B. B. Signalling pathways of the TNF superfamily: a double-edged sword. Nat. Rev. Immunol. 3, 745–756 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Wang, H., Luo, X. & Leighton, J. Extracellular matrix and integrins in embryonic stem cell differentiation. Biochem. Insights 8, 15–21 (2015).

    PubMed  PubMed Central  Google Scholar 

  61. Pathan-Chhatbar, S. et al. Direct Regulation of the T Cell Antigen Receptor’s Activity by Cholesterol. Front. Cell Dev. Biol. 8, 1728 (2021).

    Article  Google Scholar 

  62. Goyette, J., Nieves, D. J., Ma, Y. & Gaus, K. How does T cell receptor clustering impact on signal transduction? J. Cell Sci. 132, jcs226423 (2019).

    Article  CAS  PubMed  Google Scholar 

  63. Nassereddine, A. et al. Ligand nanocluster array enables artificial-intelligence-based detection of hidden features in T-cell architecture. Nano Lett. 21, 5606–5613 (2021).

    Article  CAS  PubMed  Google Scholar 

  64. Li, M. & Yu, Y. Innate immune receptor clustering and its role in immune regulation. J. Cell Sci. 134, jcs249318 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Inoue, M. & Shinohara, M. L. Clustering of pattern recognition receptors for fungal detection. PLoS Pathog. 10, e1003873 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Gay, N. J., Symmons, M. F., Gangloff, M. & Bryant, C. E. Assembly and localization of Toll-like receptor signalling complexes. Nat. Rev. Immunol. 14, 546–558 (2014).

    Article  CAS  PubMed  Google Scholar 

  67. Ma, Z. et al. Membrane nanodomains modulate formin condensation for actin remodeling in Arabidopsis innate immune responses. Plant Cell 34, 374–394 (2022).

    Article  PubMed  Google Scholar 

  68. Yap, L., Tay, H. G., Nguyen, M. T. X., Tjin, M. S. & Tryggvason, K. Laminins in cellular differentiation. Trends Cell Biol. 29, 987–1000 (2019).

    Article  CAS  PubMed  Google Scholar 

  69. Ivetic, A., Green, H. L. H. & Hart, S. J. L-selectin: a major regulator of leukocyte adhesion, migration and signaling. Front. Immunol. 10, 1068 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Mattila, P. E., Green, C. E., Schaff, U., Simon, S. I. & Walcheck, B. Cytoskeletal interactions regulate inducible L-selectin clustering. Am. J. Physiol. Cell Physiol. 289, C323–C332 (2005).

    Article  CAS  PubMed  Google Scholar 

  71. Liu, S. & Kiick, K. Architecture effects on L-selectin shedding induced by polypeptide-based multivalent ligands. Polym. Chem. 2, 1513–1522 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Li, W. et al. Large-scale topographical screen for investigation of physical neural-guidance cues. Sci. Rep. 5, 1–8 (2015).

    Google Scholar 

  73. Sun, Y. et al. Self-organizing circuit assembly through spatiotemporally coordinated neuronal migration within geometric constraints. PLoS ONE 6, e28156 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Umeshima, H. et al. Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration. Neurosci. Res. 142, 38–48 (2019).

    Article  CAS  PubMed  Google Scholar 

  75. Martinez-Garay, I. Molecular mechanisms of cadherin function during cortical migration. Front. Cell Dev. Biol. 8, 964 (2020).

    Article  Google Scholar 

  76. Shikanai, M., Nakajima, K. & Kawauchi, T. N-cadherin regulates radial glial fiber-dependent migration of cortical locomoting neurons. Commun. Integr. Biol. 4, 326 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Fujioka, T. et al. β1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain. EBioMedicine 16, 195–203 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Chronopoulos, A. et al. Syndecan-4 tunes cell mechanics by activating the kindlin–integrin–RhoA pathway. Nat. Mater. 19, 669–678 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Couchman, J. R. Syndecans: proteoglycan regulators of cell-surface microdomains? Nat. Rev. Mol. Cell Biol. 4, 926–938 (2003).

    Article  CAS  PubMed  Google Scholar 

  80. Manon-Jensen, T., Itoh, Y. & Couchman, J. R. Proteoglycans in health and disease: the multiple roles of syndecan shedding. FEBS J. 277, 3876–3889 (2010).

    Article  CAS  PubMed  Google Scholar 

  81. Sarker, F. A., Prior, V. G., Bax, S. & O’Neill, G. M. Forcing a growth factor response—tissue-stiffness modulation of integrin signaling and crosstalk with growth factor receptors. J. Cell Sci. 133, jcs242461 (2020).

    Article  CAS  PubMed  Google Scholar 

  82. Schwartz, M. A. & Ginsberg, M. H. Networks and crosstalk: integrin signalling spreads. Nat. Cell Biol. 4, E65–E68 (2002).

    Article  CAS  PubMed  Google Scholar 

  83. Nair, A., Chauhan, P., Saha, B. & Kubatzky, K. F. Conceptual evolution of cell signaling. Int. J. Mol. Sci. 20, 3292 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  84. Alberts, B. et al. General Principles of Cell Communication. In Molecular Biology of the Cell (Garland Science, 2002).

  85. Cai, D. et al. Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157, 1146 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Dan, L., Jian, D., Na, L. & Xiaozhong, W. Crosstalk between EGFR and integrin affects invasion and proliferation of gastric cancer cell line, SGC7901. Onco Targets Ther. 5, 271 (2012).

    PubMed  PubMed Central  Google Scholar 

  87. Wee, P. & Wang, Z. Epidermal growth factor receptor cell proliferation signaling pathways. Cancers 9, 52 (2017).

    Article  PubMed Central  Google Scholar 

  88. Cherkouk, C. et al. Controlled immobilization of His-tagged proteins for protein–ligand interaction experiments using Ni2+-NTA layer on glass surfaces. Clin. Hemorheol. Microcirc. 61, 523–539 (2015).

    Article  CAS  PubMed  Google Scholar 

  89. Biswas, K. H. et al. E-cadherin junction formation involves an active kinetic nucleation process. Proc. Natl Acad. Sci. USA 112, 10932–10937 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Zhang, Y., Ge, C., Zhu, C. & Salaita, K. DNA-based digital tension probes reveal integrin forces during early cell adhesion. Nat. Commun. 5, 1–10 (2014).

    Article  Google Scholar 

  91. Brockman, J. M. et al. Live-cell super-resolved PAINT imaging of piconewton cellular traction forces. Nat. Methods 17, 1018–1024 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. WC, L., CH, Y., S, T. & JT, G. Supported membrane formation, characterization, functionalization, and patterning for application in biological science and technology. Curr. Protoc. Chem. Biol. 2, 235–269 (2010).

    Article  Google Scholar 

  93. Pfaff, M. et al. Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins. J. Biol. Chem. 269, 20233–20238 (1994).

    Article  CAS  PubMed  Google Scholar 

  94. Ferhan, A. R. et al. Solvent-assisted preparation of supported lipid bilayers. Nat. Protoc. 14, 2091–2118 (2019).

    Article  CAS  PubMed  Google Scholar 

  95. Ulman, A. Ultrathin Organic Films: From Langmuir-Blodgett to Self Assembly (Academic Press, 1991).

  96. Harris, L. J. et al. The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 360, 369–372 (1992).

    Article  CAS  PubMed  Google Scholar 

  97. Xia, S. et al. Nanoscale architecture of the cortical actin cytoskeleton in embryonic stem cells. Cell Rep. 28, 1251–1267.e7 (2019).

    Article  CAS  PubMed  Google Scholar 

  98. van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011).

    Article  PubMed  Google Scholar 

  99. Cai, Y. et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys. J. 91, 3907–3920 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Rust, M., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Gauthier, N. C., Fardin, M. A., Roca-Cusachs, P. & Sheetz, M. P. Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading. Proc. Natl Acad. Sci. USA 108, 14467–14472 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Hu, W., Sarveswaran, K., Lieberman, M. & Bernstein, G. H. Sub-10 nm electron beam lithography using cold development of poly(methylmethacrylate). J. Vac. Sci. Technol. B 22, 1711–1716 (2004).

    Article  CAS  Google Scholar 

  103. Shroff, H., White, H. & Betzig, E. Photoactivated localization microscopy (PALM) of adhesion complexes. Curr. Protoc. Cell Biol. 58, 4–21 (2013).

    Article  Google Scholar 

  104. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  PubMed  Google Scholar 

  105. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Giannone, G. et al. Periodic lamellipodial contractions correlate with rearward actin waves. Cell 116, 431–443 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank G. Grenci (Nano and Microfabrication Core, MBI, Singapore) for useful discussions for cleaning nanopatterned coverslips, D. Pitta de Araujo (Science Communication Core, MBI, Singapore) for helpful suggestions for illustrations and A. Wong (Science Communication Core, MBI, Singapore) for his comments on the manuscript text. We thank the Michael W. Davidson group, The Florida State University, Tallahassee, FL, USA for the mPA-GFP-paxillin DNA construct. This work was supported by intramural funds from the Mechanobiology Institute. K.J. was supported by Mechanobiology Institute Graduate Scholarship. M.P.S. received National Institutes of Health (NIH) grant support related to this project (no. RO1-GM113022). K.J. and P.K. acknowledge funding support from Ministry of Education Academic Research Fund Tier2 (MOE2019-T2-2-014). This work was performed, in part, at the Center for Nanoscale Materials, a U.S. Department of Energy Office of Science User Facility, and supported by the US Department of Energy, Office of Science, under contract no. DE-AC02-06CH11357.

Author information

Authors and Affiliations

  1. Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    Kashish Jain, Pakorn Kanchanawong, Michael P. Sheetz & Rishita Changede

  2. Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore

    Pakorn Kanchanawong

  3. Molecular Mechanomedicine Program, Biochemistry and Molecular Biology Department, University of Texas Medical Branch, Galveston, TX, USA

    Michael P. Sheetz

  4. Center for Nanoscale Materials, Argonne National Laboratory, Lemont, IL, USA

    Xianjing Zhou

  5. Tech4Health Institute and Department of Radiology, NYU Langone Health, New York, NY, USA

    Haogang Cai

  6. TeOra Pte. Ltd, Singapore, Singapore

    Rishita Changede

Authors
  1. Kashish Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pakorn Kanchanawong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael P. Sheetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xianjing Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Haogang Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rishita Changede
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.C. developed the functionalization of Ti substrates. K.J. and R.C. designed the experiments. K.J. performed experiments, analyzed and summarized the data, illustrations and figures. H.C. and X.Z. developed Ti nanofabrication processes. The manuscript was prepared with input from all authors.

Corresponding authors

Correspondence to Haogang Cai or Rishita Changede.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Nicholas Kurniawan, Cas van der Putten, Carsten Schulte and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

Changede, R. et al. Nat. Mater. 18, 1366–1375 (2019): https://doi.org/10.1038/s41563-019-0460-y

Extended data

Extended Data Fig. 1 TIRF calibration.

Fluorescence images of a cell expressing paxillin-EGFP imaged in an epifluorescence and a TIRF mode.

Supplementary information

Supplementary Information

Supplementary Method 1.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jain, K., Kanchanawong, P., Sheetz, M.P. et al. Ligand functionalization of titanium nanopattern enables the analysis of cell–ligand interactions by super-resolution microscopy. Nat Protoc 17, 2275–2306 (2022). https://doi.org/10.1038/s41596-022-00717-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00717-3

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing