Avizo 8

The 3D ultrastructure of the cells was analysed by FIB/SEM tomography¹⁶ . For conventional observation (without correlations), cover glasses were removed by heating the specimen on a hot plate (105 °C). The resin blocks were then mounted on aluminium stubs, adhered using silver paste (Dotite D550; Fujikura Kasei, Tokyo, Japan), and coated with evaporated carbon. The specimen was set in FIB-SEM machinery (Quanta 3D FEG; FEI, Eindhoven, The Netherlands) and observed by SEM at 7–30 kV using a backscatter detector; the site for reconstruction was thus determined. For CLEM, the grid and cellular shape were easily recognised under this condition. Serial images from the block were acquired as described previously¹⁶ under the following conditions. Milling was performed with a gallium ion beam at 30 kV and a beam current of 1 nA. The slice pitch was set to 15 nm/step. Images were acquired at a landing energy of 2.5 keV. The milling and imaging cycle was repeated 1,000 times. Additional acquisition parameters were as follows: beam current = 51 pA, dwell time = 6 µs/pixel, image size = 2048 × 1768 pixels, and pixel size = 4.8 nm/pixel. The resulting image stacks were analysed using Avizo 8.1 (FEI, Burlington, MA, USA). Mitochondrial shapes were traced using the semimanual technique¹⁶ implemented in Avizo 8.1.

Tilt series alignment was performed using FEI Inspect 3D software (cross-correlation technique). The tomogram was reconstructed with the simultaneous iterative reconstruction technique (SIRT)^{80 (link)} over 50 iterations using FEI Inspect 3D software. Reconstructed volumes were visualized with VSG Avizo 8.1 for FEI systems software. A median filter minimized background noise, and a global threshold value was applied to segment the particles from the pore space.

Technical computed tomography was used to record the external and internal geometry of the fabricated structures. The analysis was performed using Zeiss METROTOM 1500 CT system (Carl Zeiss GmbH, Oberkohen, Germany) to enable coordinate measurements of complex geometric structures. All CT measurements were performed at resolution of 21.3 µm. Based on CT reconstruction, an analysis of geometry deviations was performed using GOM Inspect V7.5 (GOM GmbH, Braunschweig, Germany), while wall thickness analysis of the struts was performed using VG Studio Max 2.0 (Volume Graphics, Heidelberg, Germany). Additionally, porosity analysis was performed with Avizo 8.0 (FEI, Hillsboro, OR, USA).

Thirteen specimens were characterized tomographically using synchrotron radiation X-ray tomographic microscopy (SRXTM) at the X02DA TOMCAT beamline of the Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland. SRXTM data were obtained using a 20 µm LuAg:Ce scintillator, 20x objective lens (yielding 0.325 µm voxel resolution), at an energy level of 14 keV and an exposure time of 200 ms, as a series of 1501 equiangular projections while the sample is rotated through 180 degrees within the beam. Projections were post-processed and rearranged into flat- and dark-field-corrected sinograms, and reconstruction was performed on a 60-core Linux PC farm, using a highly optimized routine based on the Fourier transform method and a regridding procedure [19 (link)]. Slice data were analysed and manipulated using AVIZO 8.0 (ThermoFisher Scientific), and the illustrated figures were assembled using Adobe PHOTOSHOP CS. Figured specimens are deposited in the Geological Museum of Peking University (GMPKU), Beijing. Given that the X-rays from the synchrotron sources are monochromatic, differences in contrast in the resulting tomographic slices reflect the densities of the fossil materials they pass through [20 ].