FIB-SEM Investigation for Neurons-Electrode Interface Characterization and 3D Reconstruction

The interaction between biological cells and non-biological surfaces plays a pivotal role in influencing cell attachment, shaping long-term tissue responses, and ultimately determining the success of medical implants and biosensors[1]. The cell-chip electromechanical coupling plays a key role in ensuring a long-term and stable interaction between the neurons and the active elect of the neuroelectronic devices[1, 2]. In this work we investigated the primary cortical neurons response to the biomimetic microelectrodes designed through two-photon lithography technique. The goal strategies involved the use of the Focus Ion Beam – Scanning Electron Microscopy (FIB-SEM) assisted milling and imaging. The technique used is called “salami slicing” technique, or Slice and view; it allows to mill and to save the whole neuron without loss resolution at a nanometer length scale (about 5nm) and stable alignment. Importantly, the procedure used, the ultra-thin plasticization (UTP)[3–5], required different steps allowing the preservation of the integrity of the neuron structure. Firstly, the chemically fixation, then the heavy metal embedding by osmium and uranyl based to stain the membrane and protein; and at the end the resin embedding and polymerization. By coupling these two techniques we bypassed the substrate removal since the cut can be applied to any type of material, preserve the subcellular structures due to the plastic layer embedding, moreover we obtained the 3D reconstruction of the neurons deform around microelectrodes allowing the quantify of the cleft between the neurons and the electrodes. This was instrumental in providing a comprehensive and precise representation of complex biological structures. It allowed to visualize intricate details that might be overlooked in conventional representations. By embracing this advanced imaging technique, we aim to deepen our understanding of cell-material interactions and their implications for medical implants and biosensors, ultimately advancing the field. Through our investigations into how cell membranes engage with topographical features, such as nanoscale protrusions and invaginations, a remarkable discovery has emerged. Neurons membranes exhibit a pronounced tendency to deform inward and conform to protruding structures, while displaying limited outward deformation when encountering invaginating features. This intriguing asymmetry in membrane response significantly influences the width of the cleft between the cell membrane and the nanostructure's surface.<br/><br/>1. Mariano A, Lubrano C, Bruno U, Ausilio C, Bhupesh Dinger N, Santoro F Advances in cell-conductive polymer biointerfaces and role of the plasma membrane.<br/>2. Mariano A, Bovio CL, Criscuolo V, Santoro F (2022) Bioinspired micro- and nano-structured neural interfaces. Nanotechnology. https://doi.org/10.1088/1361-6528/ac8881<br/>3. A. Belu, J . Schnitker, S. Bertazzo, E. Neumann, D. Mayer, A. Offenhausser, Santoro F (2015) Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures. Journal of Microscopy<br/>4. Dipalo M, McGuire AF, Lou H-Y, et al (2018) Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization. Nano Lett 18:6100–6105<br/>5. Santoro F, Zhao W, Joubert L-M, et al (2017) Revealing the Cell–Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy. ACS Nano 11:8320–8328