Fiji imagej 1.49s

Fluorescently stained punches were imaged with an upright Leica SP5 confocal microscope equipped with a 1.95-mm working distance 20 × NA1.0 APO water dipping objective (Leica Microsystems GmbH, Wetzlar, Germany). Two-dimensional Z-stack images were recorded using a 488 nm Argon and a 633 nm HeNe laser with a 0.72 × 0.72 µm pixel size and 1–3 µm step size, resulting in 300–600 images per sample. Huygens Professional software (SVI, Hilversum, The Netherlands) with a theoretical point-spread function was used for de-convolution of the Z stacks, whereas three-dimensional rendering and image measurements were performed with Fiji (ImageJ 1.49s) and Amira (version 5.5.0; ThermoFisher Scientific, Waltham, USA) software [21 (link)]. Z stacks were loaded in Amira, after which we applied combined surface and volume rendering with standard settings. The total size of the Z stacks and three-dimensional renderings was 739 by 739 µm with a depth of 500 µm. Reference hematoxylin and eosin slides were positioned at a vertical side of the three-dimensional renderings, but were not directly continuous with the depicted areas in each case, depending on the site of imaging in the 3- to 4-mm-long cylindrical core.