Fig. 3: Imaging of myelinated axons through a mouse skull.

a Ex vivo specimen of a fixed mouse head. b–d Ex vivo imaging of mouse brain through the thinned skull of a 7-week-old mouse. The thickness of the thinned skull was approximately 40 µm. b Conventional OCM amplitude image of myelinated fibers in the first cortex layer at a depth of 70 µm from the upper side of the thinned skull. c Aberration-corrected CLASS amplitude image. The CLASS algorithm was individually applied to 18 × 18 subregions to correct local aberrations, and the corrected images were stitched together. The size of each subregion is ~11 × 11 µm², including overlap with adjacent areas. Microvessels (blue arrowheads) and myelinated axons (yellow arrowheads) are more clearly visible in the CLASS image. d Retrieved aberration maps for the detection pathway. The magnified map shows a representative aberration map. The number of correction modes N_c for each aberration map in d, set by the FOD, was about 3500. Color bar, phase in radians. e–h Ex vivo imaging through intact skull of an 8-week-old mouse. The thickness of the skull was about 100 µm. e 3D reconstruction of SHG imaging of an intact mouse skull. The dashed red box indicates the depth at which CLASS imaging was performed. It was located 150 µm below the upper surface of the skull. f OCM amplitude image of myelinated fibers. Myelinated fibers are almost invisible. g, h CLASS amplitude image and corresponding aberration maps, respectively. The image was analyzed as in c and d. N_c in h was about 10,000. i Experimental setup for in vivo imaging through intact mouse skull. The thickness of the skull was 125−150 µm. j OCM intensity image of myelinated fibers at a 200-µm depth from the upper surface of the skull. k CLASS intensity image (upper) and the corresponding aberration map (lower) for a 30 × 30 µm² area marked by the blue dotted box in j. l, m CLASS intensity images with their representative aberration maps for one of the 2 × 2 and 4 × 4 subregions, respectively. N_c used in aberration maps in k–m was about 3500. The skull thicknesses and imaging depths reported here are the raw axial movement of the sample stage.