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

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity, as well as axial calibration issues. In this work, we address this issue by leveraging the self-referenced characteristics of fractal-like structures, which simultaneously modulate cell growth and serve as axial calibration tools. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both 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. We then utilize these substrates as a platform for high-resolution bioimaging. As proof of concept, we cultivate human mesenchymal stem cells on these substrates, 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.