a, Low-stress silicon nitride (Si3N4) of 100 nm thickness was deposited on both sides of a silicon wafer. b, The Si3N4 thin film on the front side was patterned into 2 × 2 or 3 × 3 array of 40 × 40 microholes with each 1 μm hole spaced at 3–4 μm. c, The Si3N4 thin film on the back side was formed into a large window of 2.5 × 2.5 mm2 in the center. d, Photoresist (PR) was stripped off from both sides. e, The back side of the silicon wafer was etched to expose the microhole array in the center of the Si3N4 membrane. f, Collagen film was coated on the front side of the wafer. g, Following the injection of a drop of cell culture medium into the hollow cavity, an HDPE film was placed on the drop. h, The cell culture medium substrate was sealed by applying UV-curing optical adhesive between the back side of the wafer and a glass substrate. i, Cells were cultured on the collagen film over the microhole array followed by a graphene capping onto the cells. j, k, The front (j) and back (k) sides of the patterned substrate with a microhole array Si3N4 membrane. l, Collagen film coated on the top of the substrate. m–o, a microhole array Si3N4 membrane. To increase the success rate of obtaining SIMS images without microcracks in graphene, we fabricated four sets of 3 × 3 array of 40 × 40 μm2 microholes on a cell culture substrate (m).