Learn Everything You Need to Know about the Hitachi S 4300 SEM Manual PDF: General Information, Installation, Operation, Maintenance, and Troubleshooting
To visualize implanted cells in vitro, MSCs were pre-labeled with Hoechst 33342 (Sigma). Prior to seeding on the scaffolds and co-culturing with ASCs, MSCs were incubated with Hoechst 33342 for 2 h. Two experimental groups were designed in PLGA scaffolds: the MSCs alone group and the ASCs with MSCs co-culture group. The density of the cell suspension was adjusted to 1107 cells/mL, and the cell number of ASCs versus MSCs in the co-culture group was 1:1. The volume of cell suspension added was 10 μL per scaffold. To facilitate cell seeding, a filter paper was placed underneath the scaffold to gently draw cells into the channels. The scaffolds with seeded cells were subsequently incubated in a humidified atmosphere at 37 C for 15 min in a 24-well plate (1 scaffold per well) prior to being submerged in culture medium. The culture medium was changed every 2 d. At 3, 5, 7 and 14 d after culture, some scaffolds in each group were fixed in 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) for 2 h and subsequently maintained in 0.1 mol/L PBS that contained 30% sucrose for 48 h at 4 C. The scaffolds were embedded in optimum cutting temperature (OCT) compound at room temperature, frozen and sliced transversely at 30-μm thickness with a cryostat microtome. The differentiation of MSCs into neuron-like cells was detected via immunocytochemistry (ICC) of the MSC alone groups and co-culture groups. The morphology of the cells on the PLGA scaffolds was also examined under a cryo-scanning electron microscope (cryo-SEM, resolution-10 nm) (Hitachi S-4300).
hitachi s 4300 sem manual pdf
After surgery, the rats were kept warm and placed on beds of sawdust. To prevent infections, penicillin (50 000 Ukg-1d-1) was injected (im) for 5 d. The rat bladder was manually pressed three times daily until an autonomous reflex bladder emptying was established.
After the cells were cultured in the scaffold and at different points in time, some scaffolds were observed under a cryo-SEM. First, the PLGA scaffold with cells inside was mounted on an appropriate holder, plunge frozen in slushy nitrogen for 30 s, and subsequently transferred under vacuum onto the cool stage of the cryo-SEM preparation chamber of the Quorum 2100. After sputter-coating with gold/palladium, the specimen was visualized with a Hitachi S-4300 SEM.
The morphology of prepared porous PCL beads was observed by a field emission scanning electron microscope (FE-SEM; Model S-4300, Hitachi, Japan). The cross-sectional specimen was prepared by cutting them using a blade after being frozen in liquid nitrogen. The porosity of the PCL beads was estimated using mercury porosimetry (Poresizer 9320; Micromeritics, USA). To determine the porosity, it was assumed that the surface tension of mercury is 480 dyne/cm .
This manual probe station is typically used to measure conductivity, IV curves, and capacitance. It can be configured for many measurements and has a camera capable of recording video of moving MEMS devices.
100mm wafers - chips must use a 100mm carrier wafer with die attach via Fomblin or Stantovac vacuum oils. Mask materials SiO2, Si2N3, or Photoresist. Please see tool owner to gain permission for other hard mask materials. Available process gases: SF6 (0-100sccm), O2 (0-100sccm), C4F8 (0-100sccm), Ar (0-100sccm). NOTE: Tool configuration alternates between Niobate-only-etching and Non-Niobate-etching. Each configuration has a dedicated quartz clamp ring with extensive manual and plasma chamber cleaning occurring during the re-configuration. Generally, the configuration schedule alternates on a weekly basis; however, this regime may deviate from this depending on user requirements. Please see tool owner for details. Current configuration can be found in Coral comments for this tool (search both 'unresolved' and 'resolved' comments).
Wire bonding is used to electrically connect semiconductor devices with their packaging. The Westbond 4700E is a semiautomatic thermosonic ball-wedge wire bonder. It can be used to quickly lay down a succession of shaped electrical interconnects, or simple stud bumps for bonding chips using the flip chipper. The shaping and bonding of the wire is automated while the X-Y placement is manual, making it ideal for quickly generating testable prototypes and small batches of working devices. The wire is 1 mil (25um) gold wire.
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High-resolution 3D imaging is required to fully detail the intracellular habitat of the mycobacteria, but many cellular structures of interest are beyond the resolution of light microscopes. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is a valuable and little explored alternative, with significantly reduced manual labor compared to non-automated 3D electron microscopy (EM) techniques. During FIB/SEM tomography, a dualbeam instrument comprising a FIB and SEM is used to successively mill away thin slices of material (typically 10-100nm thick) from a sample block, and image the appearing sample surface with the scanning electron beam. This results in an ultra-high resolution image stack representing the cell or region of interest in three dimensions.
To meet the need for a 3D CLEM platform optimized for LM and FIB/SEM tomography, we developed a reproducible system allowing targeted high throughput and high quality FIB/SEM tomography studies of adherent cells such as macrophages. The presented system is based on a micropatterned Aclar substrate, on which adherent cells are cultured within microwells of a predefined size. The cells can be studied with light microscopy within the wells, and an integrated reference system ensures proper cell localization. After contrasting for EM and plastic embedding, the aclar substrate is removed, leaving cells embedded in small protruding epoxy blocks. We show the successful use of this system for investigation of mycobacterium-infected primary human macrophages. Cells imaged using confocal microscopy were easily relocated for 3D imaging with FIB/SEM, allowing identification of the labeled structures with significantly enhanced resolution. The sample geometry allowed for straightforward overlay between data sets from confocal and FIB/SEM, with minimum manual adjustments. Important cellular structures such as mycobacterium-containing phagosomes were accurately resolved during FIB/SEM imaging. The presented system is simple to produce and use, and increases the quality, specificity and throughput of correlative 3D confocal fluorescence microscopy and FIB/SEM tomography studies.
3D models of an M. avium infected macrophage were created from the image stacks collected with both confocal and FIB/SEM techniques, using Avizo. To create 3D models from the FIB/SEM tomography image stack, contours with increased contrast (typically lipid membranes) of structures of interest were manually traced on each image of the stack. A surface was generated from the traced contours by interpolating over the known distance between each image (i.e. 35 nm in this case) throughout the whole stack (Figs 5A and 6). Clearly resolved membranes were used to determine boundaries and internal structures of organelles, bacteria and phagosomes. The process of generating surfaces from the FIB/SEM stack is also illustrated in S1 Video. 3D surfaces were rendered from the confocal image stack was surface rendered by a threshold algorithm.