Ld plan neofluar 5x 0.15na objective

To prepare samples for AFM, the dorsal telencephalon was dissected from the embryonic brain in 1X DMEM-N2 medium (ThermoFisher) to expose the ventricular surface. Tissues were positioned with the VZ surface upward and mounted onto 50 mm glass-bottom Fluorodish (World Precision Instruments) coated with Cell Tak tissue adhesive (Corning). Tissues were then covered with 1X DMEM-N2 medium and recovered in a 5% CO₂ chamber at 37°C for 1 hour. Stiffness measurement was performed by MFP-3D-BIO AFM (Asylum Research). An Axio Observer Z1 inverted microscope (Zeiss) served as the AFM base (LD Plan-Neofluar 5x 0.15NA objective) to locate the sample and position the cantilever tip over the sample. A CP-CONT-BSG-C (sQube) probe with a 20 μm borosilicate glass bead was used for all measurements. The Asylum Research GetReal calibration method was utilized for the determination of the spring constant (~0.2 N/m). The trigger point was set to 10 nN with an approach and retraction velocity of 5 μm/sec. To determine the Young’s Modulus, the force-indentation curves were fit to the Hertz model for spherical tips through the Asylum Research Software (version 16), with an assumed Poisson’s ratio value of 0.45 for the sample ^{57 (link)}. Three distinct spots (40 x 40 μm² by size) were measured for each piece of tissue. Average stiffness of each spot was calculated for data analysis.