Nanozoomer 2.0 ht c9600 13

Animals were sacrificed by cervical dislocation. The testis and epididymis were dissected out en bloc, fixed in Bouin’s solution overnight, dehydrated in a graded ethanol series, and embedded in paraffin. Five-µm-thick serial sections with intervals of 50 µm were cut using a microtome and mounted on glass slides. Sections were treated with PAS-H to stain the basement membrane of seminiferous tubules, as previously described^{35 (link)}. Sections were digitized using a whole-slide scanner (Nanozoomer 2.0-HT C9600-13; Hamamatsu Photonics, Hamamatsu, Japan) with a 20-fold objective lens, and the resulting digital images of the sections were visualized with viewer software (NDP.view2 U12388-01; Hamamatsu Photonics).

We decided to use a single, virtual plane of focus throughout the physical glass slide. This method proved to be more difficult in maintaining a good focus throughout the slide than initially assumed. The autofocus functionality only provided a rough starting point resulting in the need to adjust the focal points in nearly every glass slide to achieve optimal results.
The slides were converted into image files with the slide scanner NanoZoomer 2.0-HT (C9600-13) (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) producing high-quality scans. Unfortunately, the scanner software did not produce an open image format that we could use. This resulted in the necessity of converting the image files into a format we could utilize. However, in the meantime a set of tools were published to handle the Hamamatsu image format (NDPI) [19 ,20 ]. We decided to implement the Zoomify file format.

Slides were digitized in brightfield at 20× magnification using the NanoZoomer 2.0-HT C9600-13 (Hamamatsu Photonics, K.K., Japan), a high-speed and high-resolution digital slide scanner system. Digital images were acquired in .ndpi format and visualized with NDP.view software (Olympus) on a standard PC.