Crossbeam 550 fib sem

For STEM characterization, first, a TEM lamella was prepared by using a Zeiss Crossbeam 550 FIB-SEM. Then, the characterization was conducted on a probe and image-corrected FEI Titan Themis Z microscope equipped with a hot-field emission gun working at 300 kV.

Specimens were loaded into the Zeiss Crossbeam 550 FIB-SEM. To locate the cell of interest, an accelerating voltage
of 1.5kV or 3kV was used with a beam current of 1.0nA and an SE2 detector. The raised gridded patterns were visible, as were the heavy metal-stained cells. Previously acquired light microscopy images identified target cells of interest. An ATLAS3D (Fibics Inc., Ottawa, Canada) sample preparation workflow was done. A protective platinum pad was overlaid after which tracking and focus lines were milled into the platinum surface and covered by a carbon deposition. A coarse trench and polish mill at 30nA and 3nA, respectively, exposed the cell on the trench face, and an acquisition run was executed. A typical run generated ~ 2000 two dimensional (2D) images.
FIB-SEM reconstructions were analyzed using IMOD (version 4.7) and 3DSlicer (version 4.6) software. The slicer module in IMOD captured 2D images in acquisition and arbitrary planes. ImageJ, iMovie, and Wondershare Filmora generated movies from the respective sections. Segmentation assignments were aided by checking the accuracy of structures in all three planes (xyz) for FIB-SEM image planes. A 3DSlicer was used for visualization and generation of merged FIB-SEM/3D segmentation images.

XRD (DX−2700B) was used to reveal the phase composition of the BiCuSeO, BiCuSe_1.05O_0.95, Bi_0.94Pb_0.06CuSeO, and Bi_0.94Pb_0.06Cu_1−xSe_1.05O_0.95 (x = 0, 0.01, 0.03, and 0.05) samples with Cu K_α radiation in a 2θ range of 10° to 80° with a step of 0.04°. Transmission electron microscope (TEM, JEOL-F200), aberration-corrected transmission electron microscope (TEM, JEOL-NEOARM 200F), EDS, and BSE imaging (ZEISS Crossbeam 550 FIB-SEM) were used to reveal the phase composition of the samples. Transmission electron microscope samples for TEM characterization were prepared using grinding, polishing, and argon ion thinning in the liquid nitrogen stage with the Ion Beam Milling System (GATAN PIPS II 695). It should be noted that the use of lightweight argon ion in the liquid nitrogen cold stage for sample thinning can effectively minimize the damage to the sample and avoid ion bombardment-induced dislocations. The IFFT image was obtained by processing the HRTEM image in the Gatan Digital Micrograph (GMS-3) software.

High-resolution SEM images of the samples deposited on silicon wafers were obtained in a Hitachi S4800 microscope at an acceleration voltage of 2 kV. Cross sectional views were obtained by cleaving the Si (100) substrates. FIB-3D was performed on a Zeiss crossbeam 550 FIB-SEM. TEM images were obtained in a CM20 apparatus from Philips. HAADF-STEM images were acquired in a Tecnai G2 F30 S-Twin STEM and a Titan Themis from Thermo Fisher Scientific (formerly FEI). Electron tomography was performed in a Titan Themis using a Fischione tomography holder. XPS experiments were performed in a Phoibos 100 DLD x-ray spectrometer from SPECS. The spectra were collected in the pass energy constant mode at a value of 50 eV using magnesium and aluminum X-ray sources. C1s signal at 284.5 eV was utilized for calibration of the binding energy (BE) in the spectra. The assignment of the BE to the different elements in the spectra corresponds to the data in Briseno et al. (2008b (link)). UV-Vis transmission spectra of samples deposited on fused silica slides were recorded in a Cary 100 spectrophotometer in the range from 190 to 900 nm.

The Autogrids were mounted onto a ZEISS-customized shuttle and transferred to a Crossbeam 550 FIB-SEM (ZEISS Microscopy, Oberkochen, Germany) using a QUORUM PP3010Z transfer system. Throughout the procedure, the samples were kept below − 170 °C. The cryo-stage was tilted to 8° to produce lamellae at a shallow angle (18° relative to the grid plane). A section surface of 80 μm in width was milled by cryoFIB with the following settings: 30 kV acceleration voltage, 5–7 nA ion beam currents. The cryoFIB milling was continuously performed on the side facing to the electron beam. The newly exposed surface was simultaneously imaged by CSEI using the in-lens and in-chamber detectors at 3 kV acceleration voltage. The SEM imaging settings included an electron beam current of 50 pA, image size of 1024 × 768 pixels, dwell time of 1.8 μs (scan speed of 5), and repetitive scans of 20 times. The in-lens/in-chamber mixed detection was performed using the smartSEM (ZEISS Microscopy) with a mixing ratio between 0.5 and 0.7. The FIB milling was stopped immediately once the target was found. Further milling should be performed only on the other side.

The crystal structure of powders samples was detected by X‐ray diffraction (XRD, Rigaku Smartlab). The chemical elements and surface bonding of MXene/PEI fibrous network were performed by X‐ray photoelectron spectrometer (XPS, ESCALAB 250Xi). The morphology of MXene nanosheets was examined by transmission electron microscopy (TEM, Talos F200S). The microstructure of MXene nanosheets, ultrathin PEI network and MXene/PEI fibrous network were observed with scanning electron microscopy (SEM, JSM 7610F Plus). The thickness of MXene nanosheet was characterized using atomic force microscopy (AFM, Cypher ES) in a standard tapping mode. The morphology of the MXene/PEI fibrous network was characterized by 3D optical profiler (Nanovea ST400). The in situ SEM for press‐release dynamic process was tested by using Zeiss Crossbeam 550 FIB‐SEM.

Mature sperm and testicular tissue were washed with PBS, fixed in 3% glutaraldehyde at 4°C overnight, then washed with 0.1 M cacodylate buffer followed by fixation with 2% osmium tetroxide at 4°C for 2h. After dehydration and processing samples were embedded in Epon C (70°C, 48 h). Ultra-thin sections were contrasted with uranyl acetate and lead citrate and then examined and imaged with a scanning electron microscope (Crossbeam 550 FIB SEM, Zeiss, Germany) equipped with a retractable STEM detector in the Microscopy Core Facility of the University of Bonn.