Lambda 900 uv vis spectrophotometer

We measured the chlorophyll content of each leaf sample spectrophotometrically^{57 (link)}. A fully expanded, well-exposed fourth true leaf of M82 and IL5-4-5-44 was cut off, and the primary and first secondary vein were carefully removed using a scalpel. The weighed samples (0.2 g) were immediately transferred to a 10-mL centrifuge tube containing 8 mL anhydrous ethanol to extract the chlorophyll, and they were stored in a refrigerator at 4 °C for 48 h. We performed all determinations in triplicate. Subsequently, the samples were centrifuged at 8000 r/min for 5 min at 4 °C. The supernatant of the chlorophyll solution was placed in a cuvette for quantification of the chlorophyll content at 665 nm and 649 nm with a Lambda 900 UV/VIS spectrophotometer (PerkinElmer Inc., Waltham, MA, USA). The entire process of leaf chlorophyll extraction was conducted in a darkroom to avoid chlorophyll decomposition. We calculated the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in the leaves using the formulas modified by Lichtenthaler (Lichtenthaler, 1987). The content of Chl a (μg/mL) = (13.95OD₆₆₅-6.88OD₆₄₉) × V/1000 W; the content of Chl b (μg/mL) = (24.96OD₆₄₉ − 7.32 OD₆₆₅) × V/1000 W. The OD₆₆₅ and OD₆₄₉ values are the absorbances at wavelengths of 665 and 649 nm, respectively. V (mL) is the volume of each sample solution, and W (g) is the fresh weight of each sample.

Morphological characterisation was performed using a field-emission scanning electron microscope (FESEM, Hitachi S-4800) with a 1.5 kV accelerating voltage. The thickness was obtained from sample cross sections fabricated using a focused ion beam (FIB) instrument (TESCAN Lyra, Brno, Czech Republic) with a gallium source at 30 kV and 180–400 pA. 2 µm of platinum was deposited to protect the surface prior to FIB cutting. FESEM images were taken in three different areas of the films and three different measurements were carried out. The thickness was calculated by averaging the nine measurements. Reflectance spectroscopy measurements of the AAO films were carried out using a PerkinElmer Lambda 900 UV–vis spectrophotometer, ranging from 200 to 900 nm using the diffuse mode with integrating sphere. CIE Yxy values, which represent a colour model including luminance (Y) and chromaticity (xy) using the CIE 1931 colour space [12 (link)], were obtained from the UV–vis reflectance measurements of the nanostructures according to the detailed description in [23 (link)]. For these calculations, only the wavelength range of 400–700 nm was considered.