3.3. CaOx Crystals Measurements in the Leaves

Leaf samples from the four C. quitensis populations were collected for the CaOx crystal projected area measurements. The collected leaves were bleached in sodium hypochlorite solution (5% p/p) in accordance with Gómez-Espinoza et al. (2020) [11]. Whole mature leaves were briefly put in an aqueous solution of commercial bleach for 48 h until full depigmentation. Depigmented leaves were rinsed with abundant distillated water and then put between two microscope slides (necessary to exert pressure and fully expand the leaves). Samples were observed under a light microscope adapted with a polarizing filter at 10× magnification using a Leica DM750-Camera and Leica ICC50W (Leica Microsystems, Wetzlar, Hesse, Germany). Several images were taken per leaf, covering the total leaf area. The area of each crystal was calculated by digital image analysis (ImageJ-Fiji v 2.0.0-rc69/1.52i) [39]. For each individual leaf, several images were taken comprising the total leaf area. Each individual image was analyzed as follows: (1) the image was converted to 8 bits, (2) the 8-bit image was converted to Mask, and (3) the tool “Analyzing Particles” was used to count and measure the area of the crystals in the picture using the following parameters: Size 400–5000 pixel ², Circularity 0.35–1.00. The total counts (crystals area) of all images from an individual leaf were added together to obtain the total area of crystals per leaf, after which this value was divided by the total leaf area to obtain a ratio area of crystals/area leaf. At least five cushions per population were used to collect leaves (n). For each cushion 10 leaves were measured.