Cells communicate with their environment via surface receptors, but nanoscopic receptor organization with respect to complex cell surface morphology remains unclear. This is mainly due to a lack of accessible, robust and high-resolution methods. Here, we present an approach for mapping the topography of receptors at the cell surface with nanometer precision. The method involves coating glass coverslips with glycine, which preserves the fine membrane morphology while allowing immobilized cells to be positioned close to the optical surface. We developed an advanced and simplified algorithm for the analysis of single-molecule localization data acquired in a biplane detection scheme. These advancements enable direct and quantitative mapping of protein distribution on ruffled plasma membranes with near isotropic 3D nanometer resolution. As demonstrated successfully for CD4 and CD45 receptors, the described workflow is a straightforward quantitative technique to study molecules and their interactions at the complex surface nanomorphology of differentiated metazoan cells.

Abbe Center of Photonics Friedrich Schiller University Jena Jena Germany

Department of Biophysical Chemistry J Heyrovsky Institute of Physical Chemistry Czech Academy of Sciences Prague Czech Republic

Department of Biotechnology and Biophysics Biocenter University of Würzburg Würzburg Germany

Department of Electrochemical Materials J Heyrovsky Institute of Physical Chemistry Czech Academy of Sciences Prague Czech Republic

Department of Physics SUPA University of Strathclyde Glasgow UK

Institute of Applied Optics and Biophysics Friedrich Schiller University Jena Jena Germany

Jena Center for Soft Matter Friedrich Schiller University Jena Jena Germany

Laboratory of Leukocyte Signalling Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

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Cell surface morphology mimicking nano-bio platform for immune cell stimulation

Role of Lipids in Morphogenesis of T-Cell Microvilli