Ion milling and scanning electron microscopy image acquisition were performed in a dual-beam scanning electron microscope (FEI Helios NanoLab 660 G3 UC). The target ROIs were localized by finder-grid coordinates (registered during optical microscopy imaging and etched to the surface of the resin block) visible under the Everhart-Thornley detector at 0.8 nA and 20 kV. On the top of a localized target cell, a protective layer of platinum (1000 nm) was deposited on top of the ROI using a single gas injection system, and a trench around the ROI was milled by a focused ion beam at 21 nA and 30 kV. FIB-scanning electron microscopy data collection was done by fine FIB milling at 0.79 nA and 30 kV (5-nm slice thickness), followed by scanning electron microscopy image acquisition at 0.2 nA and 2 kV using the In-Column backscattered electron detector with a pixel size of 3 nm and a pixel dwell time of 15 μs. The size (number of pixels) of acquisition area was adjusted to the size of the area of interest.
Helios nanolab 660 g3 uc
Manufactured by Thermo Fisher Scientific
The Helios NanoLab 660 G3 UC is a high-performance scanning electron microscope (SEM) designed for advanced materials analysis and characterization. It offers a combination of high resolution, low accelerating voltage, and versatile imaging capabilities to support a wide range of applications.
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4 protocols using helios nanolab 660 g3 uc
1
Correlative Microscopy for Ultrastructural Analysis
2
Ultrastructural Analysis of Hippocampal Synapses
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Ion milling and image acquisition was performed in Dual beam system FEI Helios NanoLab 660 G3 UC.
By using a low magnification and secondary electron imaging (20 kV and 0.8 nA) the block was oriented into best position and the region of interest was chosen. The protective layer (~1 µm thick) of platinum was deposited, using the gas injection system of the microscope, onto the surface of the block, above the region of interest. A large trench around protective region was milled at a current of 21 nA and 30 kV by focus ion beam. It was followed by fine milling at 0.79 nA and 30 kV, thickness of slices was 90 nm. The SEM imaging of the milled face (area of interest) was done in backscattered imaging mode and the serial SEM images were acquired at 2 kV and 0.2 nA using an InColumn backscattered electron detector (ICD). The region of interest was the dendritic layer below hippocampal CA1 area, determined based on preview of the low magnification images. 66 images of 9216 by 6144 nm (image 3072 × 2048 pixels, the XY pixel size was set to 3 nm) were analyzed per time point (37 °C, 16 °C–0 h, 37 °C–24 h) at each group (saline, ChABC).
Synapses were counted using FIJI (ImageJ) when: a pre- and postsynaptic density was present and vesicles in the presynaptic area detected.
By using a low magnification and secondary electron imaging (20 kV and 0.8 nA) the block was oriented into best position and the region of interest was chosen. The protective layer (~1 µm thick) of platinum was deposited, using the gas injection system of the microscope, onto the surface of the block, above the region of interest. A large trench around protective region was milled at a current of 21 nA and 30 kV by focus ion beam. It was followed by fine milling at 0.79 nA and 30 kV, thickness of slices was 90 nm. The SEM imaging of the milled face (area of interest) was done in backscattered imaging mode and the serial SEM images were acquired at 2 kV and 0.2 nA using an InColumn backscattered electron detector (ICD). The region of interest was the dendritic layer below hippocampal CA1 area, determined based on preview of the low magnification images. 66 images of 9216 by 6144 nm (image 3072 × 2048 pixels, the XY pixel size was set to 3 nm) were analyzed per time point (37 °C, 16 °C–0 h, 37 °C–24 h) at each group (saline, ChABC).
Synapses were counted using FIJI (ImageJ) when: a pre- and postsynaptic density was present and vesicles in the presynaptic area detected.
3
High-Pressure Freezing and FIB-SEM Imaging
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Cells were grown on gridded sapphire discs coated with poly-L -lysine for 2 h. Samples were immediately frozen on a Leica EM HPM100 high-pressure freezing machine. Freeze substitution was done as recently described (Müller et al., 2021 (link)) with some modifications. Briefly, samples were substituted in acetone containing 2% osmium tetroxide, 1% uranyl acetate, 0.5% glutaraldehyde, 5% water, and 1% methanol at −90°C for 46 h. Afterward, the temperature was raised to 0°C over 15 h. At this temperature, the substitution medium was changed to pure acetone and the temperature was increased to 22°C in four 15-min steps. Between steps, the acetone was exchanged for the new one at the same temperature. Afterward, samples were incubated in 0.2% thiocarbohydrazide in 80% methanol at RT for 1 h and washed four times for 15 min in acetone, followed by 2% osmium tetroxide for 1 h, brief washing in acetone, 1% uranyl acetate in 10% methanol for another hour, and washing for 4 × 15 min in acetone. Then the samples were embedded into resin with increasing concentrations (Araldite 502/Embed 812; 25, 50, 75, and 100%), 1 h each step, and left overnight in the freshly made 100% resin. After 48 h of polymerization at 60°C, the sapphire discs were removed and samples were coated with 25 nm of the platinum and imaged by FIB-SEM (FEI Helios NanoLab 660 G3 UC).
4
Sperm Preparation for High-Resolution Microscopy
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Sperm samples stored in paraformaldehyde were centrifuged (25,000 rpm, 7 min at 25 °C) to obtain the pellet of cells, which was gently re-suspended in a fixative solution containing 3% glutaraldehyde and 1% formaldehyde in 0.1 M cacodylate buffer (freshly mixed from stock solutions) and stored for 1 h on ice. After fixation, sperm were dripped onto a poly-l -lysine-coated coverslip (freshly coated high-precision coverslips) and dried at 40 °C. The coverslips with the sperm were washed three times for 5 min in 0.1 M cacodylate buffer and dehydrated by a graded series of ethanol treatments (30, 50, 70, 90, 96 and 100%). The slides were then rinsed with acetone and processed using the Critical Point Drying method (Leica EM CPD300) with acetone89 (link). Finally, sperm smears on coverslips were placed on a sample stub using conductive carbon adhesive tape and coated with 7 nm of platinum using High Vacuum Coating System (Lecia EM ACE600). Sperm cells were imaged using an FEI Helios NanoLab 660 G3 UC. The secondary electrons were captured using Through-lens detector at 1 kV and 0.1 nA (Supplementary Fig. S4 ). The raw morphological measurements are provided in the Supplementary Table S6 .
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