35 diamond knife

Polymerized flat blocks were trimmed using 90° diamond trim tool (Diatome, Switzerland) and using 35° diamond knife (Diatome, Switzerland) mounted on Leica UC6 microtome (Leica, Austria), 70 nm sections were collected on formvar-coated slot grids (EMS, USA). For sectioning, samples were carefully oriented to obtain the relevant parts of the wing blade, indispensable for the reliable data interpretation [43 (link), 45 (link)]. TEM samples were analyzed with an FEI CM100 electron microscope operated at 80kV, equipped with TVIPS camera, piloted by EMTVIPS program. Images were collected either as single frames or stitched mosaic panels to cover larger regions of the sample. Data were processed and analyzed using Fiji, IMOD 3dmod and Photoshop programs.

For assessment of the human myelin fraction by electron microscopy, myelin purified from the white matter of subjects 1995-106 and 1996-052 was used. 75 μl of each myelin sample was mixed with 75 μl 2× concentrated fixative composed of 5% glutaraldehyde, 8% formaldehyde, and 1.0% NaCl in 100 mM phosphate buffer pH 7.3. Then, the fixed fraction was spun down and resuspended in 2% agarose Super LM (Roth, Karlsruhe, Germany). After solidification, the pellet was cut into two halves and embedded in Epon after postfixation in 2% OsO₄. Ultrathin sections across the pellet were prepared using a UC7 ultramicrotome (Leica Microsystems, Vienna, Austria) equipped with a 35° diamond knife (Diatome, Biel, Switzerland). Images were taken with a LEO912 transmission electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany) using a 2k on-axis CCD camera (TRS, Moorenweis, Germany).

Control and slc12a2b^ue58 mutant animals were prepared at 4–5 dpf by high-pressure freezing using a Leica EM ICE apparatus (Leica Microsystems). As filler, a solution of 20% PVP in E3 embryo medium was used. Freeze substitution was performed as described (Weil et al., 2019 (link)). Epon-embedded animals were cut with a 35° diamond knife (Diatome) using a UC7 ultramicrotome (Leica Microsystems). Images were obtained with a LEO912 transmission electron microscope (Carl Zeiss Microscopy) equipped with a 2k on-axis charge-coupled device (CCD) camera (TRS) between 6,500 and 10,000× with the software iTEM version 5.2 (Olympus Soft Imaging Solutions). For overviews at higher magnification, 4 to 6 images were stitched to a multi-image assembly by the iTEM software. EM micrographs were processed using Photoshop Adobe Photoshop CS6 (13.0.1) x64 (Adobe Systems).

The second halves of the explanted IOLs were sent to the Max-Planck-Institute for Polymer Research in Mainz, Germany, for further analysis including scanning electron microscopy (SEM) and energy dispersive X-Ray spectroscopy (EDS). For SEM analysis 2.5 μm cross sections through the IOL material were acquired using an ultramicrotome (UCT, Leica, Germany) and a 35° diamond knife (Diatome, Switzerland) and mounted on silicium grids. SEM examinations were carried out in low voltage (<1 kV) conditions using an SU8000 microscope (Hitachi, Japan). For a local chemical analysis EDS was performed using a Quantax 400 EDS detector (Bruker, Germany) to detect exogenous chemical elements within the IOL material.
This study solely involves laboratory analyses of IOL explants. No additional procedures on humans or animals were performed. An informed consent and ethics committee approval were therefore not required.

To prepare samples, cells were washed in PBS and then fixed in 2.5% glutaraldehyde (GA) in 200 mM HEPES pH 7.4 for 30 min at room temperature then overnight at 4°C. After several washes in 200 mM HEPES buffer, samples were processed in a Pelco Biowave Pro (Ted Pella, USA) with use of microwave energy and vacuum. Briefly, samples were fixed and stained using a reduced osmium, thiocarbohydrazine, osmium (ROTO)/en bloc lead aspartate protocol (Lerner et al., 2017 (link)). Samples destined for stereological analysis were dehydrated using an ethanol series of 50, 75, 90 and 100% then lifted from the tissue culture plastic with propylene oxide, washed four times in dry acetone and transferred to 1.5 ml microcentrifuge tubes. Samples were infiltrated with a dilution series of 50, 75 or 100% (v/v) of Ultra Bed Low Viscosity Epoxy (EMS) resin to acetone mix and centrifuged at 600 g between changes. Finally, samples were cured for a minimum of 48 h at 60°C before trimming and sectioning.
For sectioning and imaging, ultrathin sections (∼50 nm) were cut with an EM UC7 Ultramicrotome (Leica Microsystems) using an oscillating ultrasonic 35° diamond Knife (DiaTOME) at a cutting speed of 0.6 mm/sec, a frequency set by automatic mode and, a voltage of 6.0 volts. Images were acquired using a 120 kV Tecnai G2 Spirit BioTwin (FEI company) with an Orius CCD camera (Gatan Inc.)

High-pressure freezing, freeze-substitution followed by electron tomography were essentially performed as described previously (Vogl et al., 2015 (link); Jung et al., 2015a (link)). After freeze-substitution and embedding in epoxy resin (Agar 100 kit, Plano, Germany), 250 nm semithin sections for electron tomography were obtained on an Ultracut E ultramicrotome (Leica Microsystems, Germany) with a 35° diamond knife (Diatome, Switzerland). Sections were placed on 1% formvar-coated (w/v in water-free chloroform) copper 100 mesh grids (ATHENE, Plano, Germany, 3.05 mm Ø) and post-stained with UAR-EMS (Science Services, Germany) and Reynold’s lead citrate.
For electron tomography, 10 nm gold particles (British Bio Cell/Plano, Germany) were applied to both sides of the stained grids. Single tilt series at 12,000-x magnification, mainly from −60 to +60° (if only fewer angles were possible, the tomograms were only accepted for quantification if the quality was sufficient) were acquired with an 1° increment at a JEM2100 (JEOL, Germany)) transmission electron microscope at 200 kV using the Serial-EM software (Mastronarde, 2005 (link)). The tomograms were generated using the IMOD package etomo and models were generated using 3dmod (Kremer et al., 1996 (link)).