3d surface profiling and high resolution imaging for refining the florin rings and epicuticular wax crystals of Pinus koraiensis needles

3D surface profiling and high resolution imaging were performed to refine the Florin rings and epicuticular wax crystals of Pinus koraiensis needles. Needles were collected from four-year-old seedlings and air-dried for surface observations. Field emission scanning electron microscopy revealed that stomata were found on the abaxial (lower) surface of needles. Measured as ca. 40 μm long, they were largely elliptical or oval-shaped. Epicuticular wax crystals were present in the epistomatal chambers as well as on the surrounding epidermis. Rodlets were prevalently found on the stomatal bands and furrows as well as within the epistomatal chambers. The presence of wax tubules was ascertained by the distinct terminal openings at their ends. The occurrence of wax ridges was evident on the epidermis near the saw-tooth margins (nonstomatal areas). No distinct wax ridges were detected on the dewaxed needles. Raised Florin rings were distinct on the stomata. White light scanning interferometry showed that the diameter and width of stomata were ca. 44.02 ± 3.33 μm and 32.10 ± 3.30 μm, respectively. Measured from the neighboring epidermis to the stomatal aperture, the mean height of the stoma reached ca. 6.23 ± 1.28 μm. Focus variation metrology allowed measuring the mean elevation angle of the stoma, reaching ca. 41.41 ± 11.25°. This is the first report on a novel approach to the establishment of quantitative criteria of Florin ring classification by nontactile 3D surface profiling beyond the previous qualitative descriptions of Florin rings of coniferous species.