Fig 1.
Experimental flows for image detection and analysis.
The S2 microfibril angle (MFA) values of radial and tangential walls were obtained from polarization optical microscopy (POM)-based imaging (step A). Tracheid morphology in a radial file unit was extracted from fluorescence microscopy (FLM)-based imaging and image analyses (step B). Finally, the two results were integrated to simultaneously evaluate S2 MFA and tracheid morphology (step C). Details of each experimental step are explained in the main text.
Fig 2.
Example of image concatenation applied to a set of retardation images.
The reconstructed retardation image comprised 72 partly overlapping retardation images in total (9 columns × 8 rows). Each image contained 66% overlapping regions with adjacent image patches in the horizontal and vertical directions.
Fig 3.
Azimuthal angle calculation from retardation imaging, and selective radial and tangential wall extraction from azimuthal angle distributions.
(A) Definitions of azimuthal angle, and radial and tangential directions in the present experimental condition. Radial and tangential planes are colored green and yellow, respectively. (B) Azimuthal angle distribution (left, purple line) of a hexagonal-shaped tracheid (right). Tangential and radial walls are defined as pixels whose azimuthal angle is near to 45° (left: Area covered by the black curve; right: Yellow pixels, directions of yellow double-headed arrows) or the remaining parts (right: Green pixels).
Fig 4.
Examples of retardation, microfibril angle (MFA), and azimuthal angle images.
(A–C) Retardation, MFA, and azimuthal angle images of latewood tracheids. (E–G) those of earlywood tracheids. (D) and (H) Sectional views of retardation (grey solid line) and MFA values (black dotted line) along red lines drawn on latewood tracheids (A and B) and earlywood tracheids (E and F). In (D) and (H), the S2 and S1+S3 layers appear as local-intensity minima and maxima, respectively. Bars, 10 μm.
Fig 5.
Selective detection of S2 or S1+S3 layers.
(A) S2 detection and (B) S1+S3 detection in a latewood, (D) S2 detection and (E) S1+S3 detection in an earlywood. (C) and (F) are merged images of (A) and (B), and (D) and (E), respectively. Red and green dots correspond to the detected S2 (local-intensity minima) and S1+S3 (local-intensity maxima) layers’ contributions, respectively. Bars, 10 μm.
Fig 6.
Normalized intra-annual transitional behaviors of a portion of the anatomical parameters.
(A) Cell wall occupancy, (B) lumen radial diameter, and (C) tangential wall thickness, respectively. (D–F) Percentage changes of each anatomical parameter (A–C) from preceding tracheids. Solid lines and the shaded area surrounding them in each figure indicate the mean intra-annual transitions and their standard deviations, respectively. Cell no. 1 corresponds to the tracheid positioned at the most earlywood. Cell number ranges from no. 1 to no. 27 in normalized radial files.
Table 1.
Correlation coefficients between anatomical parameters and S2 microfibril angle (MFA).
Fig 7.
Comparison of normalized intra-annual transitional behaviors of anatomical parameters and S2 microfibril angle (MFA).
(A) Cell wall occupancy, (B) lumen radial diameter, (C) tangential wall thickness, (D) S2 MFA on radial walls, and (E) S2 MFA on tangential walls. In (A–C), blue solid lines and the shaded area surrounding them indicate the mean intra-annual transitions and their standard deviation, respectively. In (D) and (E), black solid lines, and gray and dark gray bands correspond to the mean intra-annual trends of S2 MFA, their standard deviations, and standard errors of the mean, respectively. Red and blue lines in (D) and (E) indicate normalized S2 MFA transitions of each radial file in the radial and tangential walls, respectively. Horizontal dotted lines indicate the positions of Cell no. 15, 18, and 25 from the left side, respectively. Cell no. 1 corresponds to the tracheid positioned at the most earlywood. Cell number ranges from no. 1 to no. 27 in normalized radial files.
Fig 8.
Results of Steel-Dwass test applied to anatomical parameters and S2 microfibril angle (MFA).
(A) Cell wall occupancy, (B) lumen radial diameter, (C) tangential wall thickness, (D) S2 MFA on radial walls, and (E) S2 MFA on tangential walls. In these matrices, yellow color cells indicate the statistically significant pairs (P < 0.05). Observation cell number is 432.