2023
bioRxiv

3D single-molecule super-resolution imaging of microfabricated fractal substrates for cell culture and self-referenced imaging

Authors:

Clément Cabriel, R. Margoth Córdova-Castro, Erwin Berenschot, Amanda Dávila-Lezama, Kirsten Pondman, Séverine Le Gac, Niels Tas, Arturo Susarrey-Arce, Ignacio Izeddin

Keywords:

SMLM, Substrate-mediated cell growth engineering, cell differentiation, 3D SiO2

Abstract:

Substrate-mediated cell growth engineering has shown increasing interest in promoting in vitro alternatives to animal experiments, as it opens new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues drive the cell phenotype are not fully understood, hence the necessity to interrogate cell behaviour at the highest resolution. However, cell 3D high-resolution optical imaging on such substrates remains challenging due to their complexity, as well as axial calibration issues. This work uses multiscale 3D SiO₂ substrates consisting of spatially arrayed octahedra features of a few micrometers to hundreds of nanometers. Through optimizations of the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as calibration tools for 3D super-resolution microscopy. We use their intrinsic multiscale and self-referenced nature to simultaneously perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. With this proof of feasibility, we then use these substrates as a platform for high-resolution bioimaging. As proof of concept, we cultivate human mesenchymal stem cells cells on them, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with 3D SMLM assessment.