Fig. 4: Longitudinal observation of the myelination processes up to cortical layer 4 through the intact skull.

a Aberration-corrected images at cortical layer 1 of the same mouse recorded at P26, P33, and P47. Each image is an MIP across a 40-μm depth range. The yellow and white arrowheads mark the newly emerged myelin segments. Blood vessels oriented along the lateral and axial directions are marked with green and blue arrowheads, respectively. Scale bar: 100 μm. b Aberration-corrected images at cortical layer 1 of a different mouse from a, taken at P33 and P74. Each image is a MIP across a 20-μm depth range. Scale bar: 30 μm. c Zoomed-in images of the myelin segments marked with white and yellow arrowheads shown in b. Images are shown at P33 and P74 as well as at P40 and P47. Scale bars: 10 μm. d Box and whisker plots of myelin segment lengths measured in the images in b with ages. Data represents the length of individual myelin segments in days P33 (n = 61), P40 (n = 54), P47 (n = 47), P74 (n = 51), where n is the number of myelin segments. The red line represents the median value and open dots are outliers. The box boundaries show the upper and lower quartiles. Whiskers represent maximum and minimum values. e Total myelin segment length per unit volume derived from three different mice with age. f Aberration-corrected images on two different postnatal days at cortical layer 4. Three examples are shown. These individual pairs were MIPs over the depths of 405–435, 455–485, and 510–540 μm (from the left). The newly emerged myelin segments were marked with yellow arrowheads, whereas the remaining unchanged ones with age were marked with blue arrowheads. Scale bar: 20 μm.