1.4 numerical aperture oil immersion objective

The fabrication platform of the MAC consists of a commercially available 2PP-based direct laser writing system (Photonic Professional, Nanoscribe GmbH, Germany) and a 5-coil electromagnetic coil setup (fig. S1) (45 ). A 63× 1.4 numerical aperture oil-immersion objective (Carl Zeiss AG, Germany) was used for 3D microprinting the MAC. A refractive index matching oil (Immersol 518 f; Carl Zeiss Microscopy GmbH) was applied onto the objective to successfully detect the SF solution–substrate interface. The laser power of the 2PP system was 50 mW, which produced a printing power of 20, 30, 40, and 50 mW at a laser intensity of 40, 60, 80, and 100%, respectively. Unless otherwise specified, the laser scanning speed was kept constant at 10,000 μm s^–1.

Hippocampal neurons were fixed for 15 min with 4% paraformaldehyde. Thereafter, cells were incubated with permeabilization and blocking solution with 0.1% Triton X-100 in HS:PBS 1:10 for 20 min. Monoclonal mouse anti-MAP2 (1:200; Santa Cruz Biotechnology, Dallas, TX, United States) antibody was incubated overnight, followed by incubation with a secondary anti-rabbit IgG conjugated with Cy3 (1:500; Jackson Immuno Research Laboratories, West Grove, PA, United States) for 2 h. All antibodies were diluted with horse serum (10%) in PBS. Samples were mounted in DAKO mounting medium (Dakocytomation, United States) and observed under a spectral confocal laser scanning microscope (LSM780, Zeiss, Germany) using a 63× 1.4 numerical aperture oil immersion objective (Zeiss, Germany) under the following conditions: for excitation we used two laser lines (488 nm, 561 nm) and emission was collected in the 490–540 nm and 569–610 nm ranges, respectively. 16-bit images were collected using a pixel time of 1.58 μs and a pixel size of 110 nm.