Histological Analysis of Tree Wood Samples

We selected two trees from our best crossdated individuals and extracted a rectangular section of wood of 1 cm width and 1 cm height covering the entire sapwood section for each sample. We next divided the aforementioned pieces into 3–5 cm long segments which were boiled for one hour to soften in a 1:3 solution of water and glycerin. Using a slide microtome (Gärtner and Nievergelt, 2010 (link)) we cut transverse histological wood sections (15–20 µm thick) for image analysis. The microsections were stained using a mix of safranin (1%) and astra blue (0.5%) solutions in order to dye the cell walls containing lignin (red) or only cellulose (blue). The sections were then fixed and permanently mounted onto glass microscope slides using a synthetic resin [EukittTM, Quick-hardening mounting medium (Sigma-Aldrich)]. Digital images were captured using an AmScope 12 MP Color CMOS Digital Eyepiece Microscope Camera installed in a light transmission microscope (Leitz, Laborlux 11, Type 020-435.028) using magnifications of 40X, 100X and 200X. We created panoramic photographs stitching together multiple overlapping images using PtGui Pro (v. 8.3.3) software. This process allowed us to compare the anatomical and sanded scanned images used previously for measuring ring-width side-by-side.