Dp73 microscope camera

Freshly moulted and inter-moult specimens of C. acosta were treated as described above, including the complete dehydration step in acetone before being embedded into Agar Low Viscosity Resin (LVR, Agar Scientific, Stanstead, Essex, UK). Cured resin blocks were sectioned at 1 μm (semithin) or 60 nm (ultrathin) on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Semithin sections were stained with toluidine blue for few seconds on a heating plate at 60 °C, whereas ultrathin sections were contrasted with uranylacetate for 30 min and leadcitrate for 5–10 min. Light microscopy sections were viewed with an Olympus BX53 equipped with a DP73 microscope camera (Olympus, Tokyo, Japan). Ultrathin sections were analyzed with a Zeiss Libra120 TEM (Zeiss, Oberkochen, Germany).

The serially sectioned specimen of Leptosynapta cf. minuta was photographed with an Olympus DP73 microscope camera mounted on an Olympus BX53 compound microscope. For the overview image stack a 10x objective (0.4 NA) was used, resulting in a pixel size of 230 nm in the sectioning plane and a pixel length of 1 µm perpendicular to the sectioning plane. A 20x objective (0.75 NA) was used for the high-resolution image stack, which lead to a pixel size of 110 nm in the sectioning plane and again a pixel length of 1 µm perpendicular to the sectioning plane due to the slice thickness. With a dry lens and based on the assumption of a mean visible light wavelength of 450 nm, the Rayleigh criterion⁵⁰ indicates a resolution in the sectioning plane of 686 nm in the overview data set and 366 nm in the high-resolution data set. The acquisition of the overview image stack, containing 1208 sections, took 2.5 h and of the high-resolution volume, containing 326 sections, took 1 h.
The image stack was converted into greyscales using Photoshop CS6 (Adobe) and adapted uniformly for better comparison with the NanoCT data. Very few missing slices where replaced by the previous or following section. For alignment, the image stack was imported into Amira (Thermo Fisher Scientific). Thereby, the ‘AlignSlices’-module was used with manual corrections of individual slices.