Quantifying Shell Mineralization via Birefringence

For each pCO₂ treatment, 5 to 8 larvae were randomly chosen among the previous subsamples and observed under polarized light to determine birefringence patterns with an Olympus dissecting microscope equipped with polarizing filters. All polarized images were acquired with an Olympus camera at ×100 magnification with 40 ms light exposition. Birefringence under polarized light is due to the mineral phase composing the shell [40] , [68] (link), [70] (link), [71] . In the absence of mineralized structures, there is no birefringence and the picture looks totally black. Under identical light conditions, areas appearing more birefringent contain a much larger proportion of crystalline calcium carbonate [70] (link), [72] (link). The intensity of birefringence of each shell was used as a proxy for mineralization level for the three pCO₂ treatments. It was quantified from pictures by using ImageJ software [69] . Pictures of polarized shells were first transformed into grayscale images. A mean gray value (in pixels) was determined for each birefringent zone. All birefringent zones of the shell were compiled to obtain a global mean gray value, giving the intensity of the birefringence of the whole shell.