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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

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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.

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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.

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.

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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.

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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

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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.

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Correspondence to Haogang Cai or Rishita Changede.

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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.

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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

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