Tecnai f20 200 kev

Resin blocks with specimens were in most cases trimmed so that the block face was perpendicular to the longitudinal axis of the worm nose for serial cross-sections, while keeping a small amount of resin around the specimen. For longitudinal images of the nose sensilla, the block face was aligned parallel to the longitudinal axis of the worm nose. Serial ultrathin sections (70-nm thickness) were collected on slot grids covered with Formvar support film, and post-stained with 2% uranyl acetate (0379, Polysciences, Inc., Warrington, PA) for 30 min, and Reynold’s lead citrate (Lead nitrate—17,900, EMS, and Sodium citrate—S-279, Fisher) for 15 min. Serial sections were imaged on a Tecnai F20 (200 keV) or F30 (300 keV) transmission electron microscope (FEI, Hillsboro, OR) and recorded using a 2K × 2K charged-coupled device (CCD) camera at 14,500X magnification on the F20 (1.25 nm pixel size), or at 23,000X magnification on the F30 (1 nm pixel size). For large overviews of the cross and longitudinal sections, we acquired montages of overlapping high-magnification images in an automated fashion using the microscope control software SerialEM (Mastronarde, 2005 (link)).

Serial sectioning, electron microscopy and tomography, and image processing were performed as described previously (Doroquez et al., 2014 (link)). Briefly, serial plastic sections (70-nm thick) were collected on slot grids covered with Formvar support film, post-stained with saturated solution of uranyl acetate (0379, Polysciences, Inc., Warrington, PA) for 15 min, and Reynold’s lead citrate (Lead nitrate - 17900, EMS, and Sodium citrate - S-279, Fisher) for 7 min, and imaged using a Tecnai F20 (200 keV) or F30 (300 keV) transmission electron microscope (FEI, Hillsboro, OR) equipped with a 2K ×2K charged-coupled device (CCD) camera. For large overviews of sections, we acquired montages of overlapping high-magnification images. For electron tomography, BSA-coated, 10 nm colloidal gold fiducials (Au - Sigma-Aldrich, St. Louis, MO; BSA - SC-2323, Santa Cruz Biotechnology, Inc.) (Iancu et al., 2006 (link)) were applied to the sections, before acquiring dual-axis tilt series with a tilt range of ±60° with 1° increments around each axis. Automated montage and tilt series acquisition was facilitated by the microscope control software SerialEM (Mastronarde, 2005 (link)). Image processing, such as blending montages and reconstructing tomograms, was performed using various tools from the IMOD software package (Kremer et al., 1996 (link)).

The precipitated minerals were collected by scraping, washed twice with 1× PBS (pH 7.4), and suspended in sodium hydroxide (10%) for 30 min. Mineral content was extracted twice with acetone or chloroform and washed twice with 100% ethanol. For X-ray diffraction, purified minerals were air-dried in a spin-vacuum for 30 min at 60 °C. The crystallinity, size, texture, and homogeneity of the dry powder were analyzed with a Bruker D8-Discover X-Ray Diffractometer. For high-resolution transmission electron microscopy, the mineral suspension (in 100% ethanol) was ground manually in a glass grinder and dropped onto a carbon-coated copper grid (Sigma–Aldrich) to allow the ethanol to evaporate. The ultrastructure was examined under a transmission electron microscope (FEI Tecnai F20 200 keV, JEM-2100F, or FEI Titan 80-3000) equipped with a field emission gun (0.1-nm lattice resolution).