For serial block-face SEM imaging, whole retinas were fixed in a mixture of 2.5% glutaraldehyde and 2% formaldehyde in sodium cacodylate buffer with 2 mM calcium chloride at RT for 5 min, and then on ice for an additional 2–3 h. Fixed retinas were stained with osmium tetroxide and uranyl acetate, and mounted and stained with lead aspartate for serial 3-D SEM as described previously (Pfeifer et al., 2015 (link)). The trimmed, resin-embedded stained blocks were imaged using a 3View serial block-face imaging system (Gatan) installed on a SIGMA-VP (variable pressure; Carl Zeiss) SEM, operating at an accelerating voltage of 1.5 kV using a standard 30-µm condenser aperture. The SEM was operated in high vacuum. Data were collected with a pixel size of 5.75 nm in the x–y plane and 25 nm along the z axis. Individual block-face electron micrographs were locally aligned with IMOD software (Kremer et al., 1996 (link)). The synaptic terminals of bipolar cells were recognized in the inner plexiform layer (IPL) by the presence of synaptic ribbons, vesicles, mitochondria, and their distinct morphology. Full 3-D reconstructions of the two bipolar terminals were made by two blind users by hand segmentation of the plasma membrane, ribbons, mitochondria, and microtubule structures of the individual serial images in Amira software (FEI) and TrakEM2 software (FIJI).
3view serial block face imaging system
Manufactured by Ametek
The 3View serial block-face imaging system is a lab equipment product that provides high-resolution, three-dimensional imaging of biological samples. The core function of this system is to capture sequential images of the exposed surface of a sample, allowing for the reconstruction of a detailed, three-dimensional representation.
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2 protocols using 3view serial block face imaging system
1
Serial Block-Face SEM Imaging of Retinal Bipolar Cells
2
Multimodal Electron Microscopy Imaging
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Liver samples mounted on aluminum pins were inserted into the specimen stage of a Sigma-VP (variable pressure) SEM (Zeiss Inc.) equipped with a 3View serial block face imaging system (Gatan Inc). The same area of a sample was imaged at the primary energies, Elow and Ehigh. The SEM was operated in high vacuum mode using a condenser aperture of diameter 30 µm. For a cutting increment of 25 nm and with a probe current of 75 pA, pixel dwell times of 3.5 and 1.5 µs per pixel were selected for imaging at primary beam energies of 1.0 keV and 1.4 keV, respectively. For a cutting increment of 50 nm and with a probe current of 48 pA, pixel dwell times of 7.0 and 3.5 µs per pixel were selected for imaging at primary beam energies of 1.4 keV and 2.2 keV, respectively. For each cut of the block face, the BSE image with lower landing energy was acquired before the image with higher landing energy. No differences in image quality or computed stain distributions were observed when the order of image acquisition was reversed. Image dwell times and pixel sizes were selected to provide appropriate values of the electron fluence that gave the highest image quality, while maintaining uniform cutting of the sample. The electron fluence on the sample block was 15 e/nm2 for 25-nm cutting increments, and 20 e/nm2 for 50-nm cutting increments.
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