Z-Axis Calibration in Optical Sectioning from Xylem Cross Sections for Grain Angle Measurement

Abstract

Optical sectioning using confocal microscopy may be problematic under some conditions due to contamination with light from outside the focal plane and resulting z-axis compression. These problems can affect quantitative wood anatomy, such as grain angle measurement. In the present report, the exact surface of xylem sections, z-axis scaling, and available scanning depth with confocal microscopy were determined in xylem transverse sections of Japanese cedar (Cryptomeria japonica D. Don). The optical section containing the sample surface was determined using power spectral analysis to find the sharpest image. Image cross-correlation analysis in serial transverse optical sections revealed that the optical sections above the sample surface showed no tangential shift with that of the surface, indicating the non-focal cell wall information. Optical sections using an oil immersion lens with oil and a dry lens without oil were compared. Optical sections with an oil lens were relatively precise while those with a dry lens showed a z-axis distortion of about ×1.5 due to the mismatch of refractive index. Therefore, the exact cell orientation angle without oil can be obtained by the two-thirds multiplication. Adequate cell wall information was available up to c. 80 μm deep.