Fig. 1. Recent developments of building BNIs at different scales. (a) and (b) Schematic of 2D lithography of BNIs; 2D BNIs fabricated on a CMOS nanoelectrode array improved the nano-bio interface to enable intracellular recording from rat neurons. The device contains a 64×64=4096 nanoelectrodes, array and records intracellular signals from more than 1700 rat neurons;¹⁷ (c) and (d) schematic of 3D additive printing of BNIs; 3D additive printing of bioengineered 3D brain-like layered structures;²⁰ and (e) and (f) schematic of 3D DLW of BNIs; DLW of 3D BNI in hydrogel material.⁴⁴

Fig. 2. Simplified diagram showing the process of 3D DLW and the TPA phenomenon.

Fig. 3. (a) Schematic of a 3D DLW set-up, including (i) the laser source (ultra-fast pulsed or CW laser); (ii) a motion system consisting of nano-piezo-stages and galvo-mirrors; (iii) beam manipulation optics, which might consist of 4-f systems, high-NA objective, etc.; (iv) beam intensity control systems, including a shutter (either an acoustic optical modulator or a mechanical shutter); and (v) a user interface to control the system with a high-performance computer. (b) Layer-by-layer DLW fabrication of a representation of the Oriental Pearl Tower of Shanghai. Right: computer-aided design (CAD) model. Left: SEM image of the fabrication result. (c) Line-by-line fabrication of biomimetic neuron structures.⁶⁷ Right: CAD model. Left: SEM image of the fabrication result.

Fig. 4. The 3D BNIs for a 3D real-time neuron migration study.⁵⁸ (a) Bright-field optical microscopic image showing the distribution of cells in a 52-μm pore-sized microstructure 5 h after seeding the cells. (b) The 3D FM image showing the 3D distribution of the cells inside a scaffold 24 h after seeding the cells. (c) Top view of the fluorescent microscope of the cells in 52-μm pore-sized scaffolds showing the non-uniform distribution of the cells under varied physical obstructions. (d) Tracking of a typical cell migration phenomenon in a 25-μm scaffold.

Fig. 5. 3D BNIs for supporting the neurite/neuron orientation and 3D network formation. (a) Left: SEM images and the CAD model of the microtowers fabricated by 3D DLW. SEM images of neuronal cells on the microtower after one week of cell culture.⁶⁰ Right: 3D reconstruction of the confocal microscopic image showing the neuronal cell distribution on the tower. The neuronal markers are MAP-2 (green), β-tubulin III (red), and DAPI (blue). (b) Left: SEM images of neuron-cages fabricated by DLW using ORMOCER^®.⁵⁹ Right: confocal microscope images of type II and type III neurocages after eight days of culturing. MAP-2 (green) and astrocytes GFAP (red) are used to stain the neuronal cells.

Fig. 6. Hybrid photoresist fabricated by 3D DLW of a 3D network-like structure for neuron force measurement, the photoresist is Ormocomp^®.⁶¹ (a) and (b) The 3D reconstruction of a confocal microscopic image of cardiomyocyte cells attached in the network-like microstructure, where the posts are connected by beams with a diameter of 0.6μm (oblique view and top view). (c) The SEM image of a fully 3D network-like microstructure for cell force measurement. The sample is labeled by F-actin (green) and A-actinin (red). (d) Video frames of the deformation process of the beam in contact with the cell, where the bending of the beam can represent the cell force during one cycle of contraction (time in seconds). (e) The experimental plot of force–beam deflection, where the cell force can be calibrated by the deformation of the beams.

Fig. 7. Modified and natural polymers fabricated by 3D DLW for 3D neuron network guidance. (a) Microstructures fabricated using PLA to support PC12 neuronal cell growth. After five days of culturing, PC12 cells proliferate in woodpile structures (300×, 700×, and 1500× magnifications are shown).⁵⁰ (b) Microstructures fabricated on an HA hydrogel surface to promote the guidance growth of hippocampal progenitors (E16).⁶² Fluorescence image of the neuronal marker b-III tubulin (green) after fixation. Scale bar, 250μm. (c) A live C. elegans worm encapsulated in a BSA structure on a photopolymerized microstructure (base).⁶³

Fig. 8. BNIs for ANNs. (a) The 3D optical interconnects fabricated by 3D DLW.⁶⁴ The interconnected structure functions as a Haar filter, which is regarded as a fundamental unit in CNNs. (b) Neuron-inspired Steiner tree low-density metastructures; the scale bar is 2μm.¹¹⁶

Fig. 9. (a) The structure of feed-forward ANNs. (b) The effective parameters in the 3D DLW process.

Fig. 10. ANNs-based adaptive optics technology for laser focusing optimization. (a) ANNs-based adaptive optics techniques enable the aberration correction of Gaussian-shaped laser beams.¹²⁵ (b) Experimental results of Kerf acquired without and with optimized focusing parameters.¹²⁶