pores were larger than the pore size (~8 mm); these protoplasts swelled out of the pores (see protoplasts indicated by arrows in Fig. 3c). This may mean that the micropores are not sufficiently large as templates for Bryopsis protoplasts. Figure 4c shows that ca. 67 % of the protoplasts were larger than the pore size. Nevertheless, 82 % of the protoplasts were 14 mm or less in diameter. On the other hand, in the case of a normal glass plate, the percentage was only 31 %. This indicates that the 10 mm pitch template can also be applied to the size control of Bryopsis protoplasts, although the 20 mm pitch template is more suitable for the present objective.As for the microstructured 7 mm pitch TiO 2 , only a small portion of the protoplasts fit into the hemispheric pores. This suggests that these micropores, with a diameter of ca. 6 mm, are too small to prepare Bryopsis protoplasts.Thus, we could successfully control the size of Bryopsis protoplasts by the use of the microstructured TiO 2 templates. The diameter of micropores should be 8 mm or larger. Among the microstructured TiO 2 templates examined, the 20 mm pitch template (pore size,~17 mm) is the template of choice.
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