Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their “life‐like state” for studying the cell interface with either 3D well‐like structures or gold‐coated mushroom‐shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide–thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell‐derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high‐quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications.

Background - Tissue-integrated micro-electronic devices for neural stimulation hold a great potential in restoring the functionality of degenerated organs, specifically, retinal prostheses, which are aimed at vision restoration. The fabrication process of 3D polymer-metal devices with high resolution and a high aspect-ratio (AR) is very complex and faces many challenges that impair its functionality. Approach - Here we describe the optimization of the fabrication process of a bio-functionalized 3D high-resolution 1mm circular subretinal implant composed of SU-8 polymer integrated with dense gold microelectrodes (23µm pitch) passivated with 3D micro-well-like structures (20µm diameter, 3µm resolution). To this end, a nickel (Ni) evaporated silicon (Si) wafer was sequentially spin-coated with SU-8 and photolithographed layer-by-layer, with a sharp electrode formation achieved through a two-step bi-layer lift-off process using LOR/AZ, followed by Cr/Au thin-layer sputter deposition to increase the adhesion. Next, the device was released by overnight Ni wet-etching using nitric acid, after which it was bio-functionalized with N2 plasma treatment and the addition of the bio-adhesion molecule arginine-glycine-aspartic acid (RGD). Main results - In-vitro and in-vivo investigations, including SEM and FIB cross section examinations, revealed a good structural design, as well as a good integration of the device in the rat sub-retinal space and cell migration into the wells. The reported process and optimization steps described here in detail can aid in the design and fabrication of similar neural implants. Conclusions - The reported process and optimization steps described here in detail can aid in the design and fabrication of retinal prosthetic devices or similar neural implants.