Fib sem system

3D rendering of synapses was reconstructed from the serial EM images of more than 500 sheets with 5 nm thickness obtained by 3VIEW focused ion beam/scanning EM (FIB/SEM) system (FEI, USA) in the FEI China-Shanghai facility, according to the manufacturer's protocol, as previously described⁵⁰ . In brief, the block face was trimmed using a sharp knife. SEM images of the untrimmed block face were used to select the desired field of view before the final trimming step, producing the desired small cut pyramid. About 800 serial images were taken using an environmental SEM Quanta FEG 200 (FEI, Netherlands) with a 3VIEW serial block face sectioning and imaging system (Gatan, USA) at an accelerating voltage of 5 keV. The equipped diamond knife used was custom made by diatome (Diatome, Switzerland), and the section thickness was 5 nm. Images were collected at an imaging size of 1 k × 1 k, pixel size of 2.47 nm, pixel dwell time of 80 μs, chamber pressure of 0.50 Torr and spot size of 3. Finally, serial images were aligned and 3D structures of more than three typical synapses in each group were rendered using an incorporated TrakEM2 plugin in combination with Imiris 8.0 software (Bitplane, Switzerland).

Elytron scales were dissected from female adult beetles to observe the surface morphology. Each specimen was fixed on a glass slide and viewed under stereoscopic microscope (SteREO Discovery V20, Zeiss, Germany) and confocal laser scanning microscope (CLSM; OLS3000, Olympus, Japan). The details of pits on elytron surface were obtained by environmental scanning electron microscope (ESEM; JEOL JSM-6700F, FEI Company, USA).
To determine the internal three-dimensional structure of the cuticle, FIB-SEM was performed using a FIB-SEM system (GAIA3, Tescan, Czech Republic). In addition, three-dimensional reconstruction was conducted by the three-dimensional visualization software Dragonfly Pro 2.0. The FIB-SEM system is equipped with a dual beam consisting of a scanning electron beam (SEM beam) and a focused ion beam (FIB). Each milling step of the ion beam was performed with 119 µA emission and 5 kV acceleration voltage. In case of possible structural damage to the cut edge by the scalpel, the middle position of the elytron surface was chosen for the tests. Serial sectioning was performed by 200 consecutive millings, each with a thickness of 30 nm. The depth of focus was 9.3 µm. The volume of each serial section was 12.7 µm × 6 µm × 9.3 µm.