5d4 camera

The isolated brain and eyes were imaged with the focus stacking method using a digital camera (Canon 5D4 camera with Canon MPE 65 mm Macro lens, Canon, Japan) mounted on the electronically-controlled focusing rack (Castel-Micro focusing rack, Novoflex, Germany). A sequence of close-up images was captured from the dorsal end of brain to the ventral end using 0.1 mm step for small samples or 0.25 mm step for large samples. Focus stacking (20–80 images) was processed using the software Helicon Focus Pro (version 7.6.4, Helicon Soft Ltd. Ukraine), rendering an image with a greater depth of field.

The isolated cuttlefish brain and eyes were processed with a standard protocol of wax histology. In brief, the samples were dehydrated in increasing concentrations of ethanol, delipidised in xylene and paraffinized in a histology processor. Sections (10–15 μm thickness) were prepared using the microtome (Leica RM2235, Germany) and stained with Haematoxyline and Eosin. The selected horizontal sections were imaged using a digital camera (Canon 5D4 camera with Canon MPE 65 mm 1-5X Macro lens at 3X magnification, Canon, Japan) mounted on the electronically-controlled focusing rack (Castel-Micro focusing rack, Novoflex, Germany). Additional processing of images for brightness adjustment was performed using Fiji (version 1.53c, open-source software, https://imagej.net/).¹⁰⁶ (link)