From the collected data sets, we identified Bowman's layer on the basis of the characteristic punctate pattern in the SHG images (
Fig. 1A).
12 As the focus moved gradually deeper, collagen lamellae appeared as fine, short, and narrow SHG linear signals and subsequently passed out of the visualized field in the continuous images. The projection of these images from the base of Bowman's layer to a depth of ∼50 μm revealed the sutural lamellae (
Fig. 1B). Three-dimensionally reconstructed projection images revealed adherence of the sutural lamellae to Bowman's layer (
Figs. 1C, D). The continuous SHG images with determined optical slices allowed the
z-axis distance between two points to be given by the number of the plane (height
a). The distance between the point at which collagen lamellae adhered to Bowman's layer and that at which the lamellae disappeared was measured (length
b). The angle (θ) of collagen lamellae adherence to Bowman's layer was thus provided by: θ = tan
–1 (height
a/length
b) (
Fig. 2). The angle of five sutural lamellae was measured for each data set, resulting in the evaluation of 15 sutural lamellae for each subject. The angle of lamellae located 50 or 100 μm below Bowman's layer was similarly measured for nine to 13 lamellae in each subject. For these measurements, we reviewed the data set around a depth of 50 or 100 μm, found collagen lamellae this depth, identified the initiation and termination points of these lamellae in the
x-y data set, measured the
x-y distance between the initiation and termination points of each lamella, and counted the number of
x-y slices. The total number of evaluated lamellae was 405, 351, and 297 for sutural lamellae, lamellae 50 μm below Bowman's layer, and lamellae 100 μm below Bowman's layer, respectively.