A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans.

Strains and growth conditions

C. elegans animals were maintained at 20°C on standard nematode growth media plates seeded with E. coli OP50 bacteria. Wild-type C. elegans (Bristol strain N2) was obtained from the Caenorhabditis Genetics Center.

Specimen preparation

Worm samples were prepared by transferring 1-day-old adult hermaphrodite worms to 20% bovine serum albumin (BSA) in M9 buffer in the cavity of an aluminum planchette (type ‘A’ hat; 100 µm deep, Wohlwend, Switzerland) for high-pressure freezing ( McDonald et al., 2007 ). In some cases, animals were treated with 10 mM levamisole and embedded in 2.4% low-melting point agarose pads ( Kolotuev et al., 2009 ). We noted that levamisole treatment and agarose embedding resulted in shortened channel cilia lacking distal segments; thus, this method was not used to model the distal ends of amphid channel cilia. The flat surface of another planchette (type ‘B’ hat) was placed on top to enclose the worms in the planchette’s cavity. The specimen–planchette sandwich was rapidly frozen using a Leica EM HPM100 high-pressure freezing system (Leica Microsystems, Vienna, Austria). Freeze-substitution was performed at low temperature (−90°C) over 3 days in a solution containing 1% osmium tetroxide (19,100, EMS), 0.5% glutaraldehyde (16,530, EMS) and 2% water in anhydrous acetone (AC32680-1000, Fisher) using a Leica EM AFS2 freeze-substitution system. The temperature was progressively increased up to 0°C (5°C/hr), and finally brought to 4°C and maintained at this temperature for 1 hr. Samples were washed four times with anhydrous acetone (30 min each), then infiltrated and embedded in Araldite 502/Embed-812 Resin (Araldite—10,900, EMS, Embed-812—14,900, EMS, DDSA—13,710, EMS) at room temperature and polymerized in an oven at 60°C for 3–4 days. Serial section TEM 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 ). Serial section ET After analyzing TEM images of the serial sections, grids, and sections with structures of interest were prepared for ssET by coating both sides of the serial sections on Formvar with 10-nm colloidal gold fiducials (Sigma-Aldrich, St. Louis, MO) that were previously incubated for 30 min in 5% BSA (SC-2323, Santa Cruz Biotechnology, Inc.) ( Iancu et al., 2006 ). Dual-axis tilt series, that is, two orthogonal tilt series of regions of interest, were acquired by tilting the sample from −60 to +60 with 1° increments using the microscope control software SerialEM ( Mastronarde, 2005 ) on a Tecnai F20 (200 keV). All images were digitally recorded on a 2K × 2K CCD camera, at 14,500X or 19,000X magnification, resulting in a pixel size of 1.25 nm or 1.04 nm, respectively.

Image processing ssTEM

Image processing to generate and analyze ssTEM 3D reconstructions was automated using manual correction only as needed. Blendmont, a utility of the IMOD software package ( Kremer et al., 1996 ) was used to finely align and stitch image tiles into a single montage image for each whole worm cross section. Images were converted from MRC to TIFF format with mrc2tif (IMOD) and then to PNG format (ImageJ) while preserving image resolution. The ir-tools ( http://www.sci.utah.edu/download/ncrtoolset ) were modified and used for automated section-to-section image registration ( Tasdizen et al., 2010 ). Nornir Build Manager software ( http://github.com/nornir ) was used to coordinate execution of the ir-tools, including the calculation of transforms for each section and the generation of the final volume with registered images ( Anderson et al., 2009 ). Briefly, volume assembly began with automatic image registration of all adjacent section pairs. The centermost section was not warped and designated as reference in the volume center. The remaining sections were mapped in relation to the center by serially applying the intervening adjacent section-to-section transforms and warped to correct for distortions resulting from the sectioning process or section folds. Where automatic registration failed between adjacent sections, the alignment was done interactively using Pyre (Nornir) or ir-tweak (ir-tools). The registered serial section images were then converted from PNG format to TIFF (ImageJ) and then to MRC format (tif2mrc/IMOD) to allow stacking of the aligned images into the ssTEM 3D reconstruction (newstack/IMOD) ( Kremer et al., 1996 ). Contours of cell membranes and other features were manually traced in IMOD to generate 3D graphical models (3dmod, imodmesh/IMOD) ( Kremer et al., 1996 ). ssET 3D tomographic reconstructions (dual axis) were generated using etomo from the IMOD software package ( Kremer et al., 1996 ; Mastronarde, 1997 ); gold fiducial markers were used for alignment of the tilt series images and the tomograms were calculated in silico using weighted back projection. Serial section tomograms were then aligned, joined, and modeled in 3D using IMOD tools.

Additional files 10.7554/eLife.01948.037 Supplementary file 1. ( A ) isMTs in amphid neuron cilia. ( B ) Location of vesicles in sensory cilia. DOI: http://dx.doi.org/10.7554/eLife.01948.037