Niu compound microscope

For histological analyses, the specimen was cut into two pieces approximately in the thorax region to ensure proper resin infiltration. The obtained pieces were afterwards dehydrated in an acidified dimethoxypropane solution, followed by several rinses in pure acetone before infiltration started in Agar Low Viscosity resin (Agar, Stansted, Essex, UK). After embedding and polymerization of the resin, ribbons of serial sections were conducted with a Diatome HistoJumbo diamond knife (Diatome, Switzerland). Sections were stained with toluidine blue, sealed and afterwards photographed and analysed with a Nikon NiU compound microscope equipped with a Nikon DsRi2 microscope camera (Nikon, Tokyo, Japan). Serial sections were converted into greyscale using FIJI^{43 (link)} and afterwards imported into the 3D visualization software Amira (Thermo Fisher). Images were aligned using the AlignSlices module of Amira. Afterwards, virtual sections of the CT stack of the parasitized and reconstructed specimen were placed using the histological sections as reference in order to compare the CT information with the histological details.

Heads of D. bosnicus and D. discolor were cut from the remaining body for histological processing. First samples were dehydrated with acidified dimethoxypropane followed by three rinses with acetone before being infiltrated and embedded in Agar LVR resin (Agar Scientific, Stansted, UK). Cure resin blocks were serially sectioned with a Diatome HistoJumbo diamond knife (Diatome, Nidau, Switzerland) at 1 µm section thickness on a Leica UC6 ultramicrotome (Leica microsystems, Wetzlar Germany). Sections were stained with 1% toluidine blue and sealed in epoxy resin. Analysis and photography of the serial sections was conducted on Nikon NiU compound microscope with a Nikon DsRi2 microscope camera (Nikon, Tokyo, Japan).
Image stacks were converted to greyscale and contrast-enhanced with F:IJI^{28 (link)} and subsequently imported into the visualization software (Thermo Fisher Scientific). Alignment of consecutive sections was conducted with the AlignSlices Tool of Amira. Structures of interest (tentorium, nervous system and digestive tract) were semi-manually reconstructed by labelling with a brush and interpolating several consecutive sections. Surfaces were calculated from the segmentation masks, followed by surface optimization using iterated smoothing and polygon-reduction steps. Snapshots were taken with the Amira software.

Prior to embedding, stored samples were postfixed in 1% aqueous osmium tetroxide (OsO₄) for 1 h and rinsed at least 3 times in double distilled water (20 min each). Osmification was followed by dehydration with acidified 2,2-Dimethoxypropane (DMP, 30 min) and rinsing in pure acetone at least 3 times (20 min each). Dehydrated specimens were then infiltrated with low viscosity resin (LVR, Agar Scientific, Stansted, Essex, UK), using acetone as intermediate (overnight). Finally, the infiltrated samples were embedded in silicone moulds. Polymerisation of the resin blocks was done at 60 °C for 12 h.
For histological investigations, ribbons of serial sections (1 µm) were produced, using a Histo Jumbo knife (Diatome, Nidau, Switzerland) mounted on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) (see [39 (link)]). Serial sections were stretched on the object slide at 70 °C and stained with toluidine blue (1%, 40 s, 60 °C). The semi-thin sections were analysed and photographed using a Nikon Ni-U compound microscope (Nikon, Tokyo, Japan) with a mounted Nikon Ds-Ri2 camera.