## 3 Results
### Imaging of Biological Samples
In our study, displacements of a tissue surface owing to breaths were measured to be \(\sim\)20 \(\upmu\)m without a sealing window and these displacements would decrease with increasing depth. However, tissue movement, an axial resolution of 2.14 \(\upmu\)m (1.58 \(\upmu\)m in tissues), and a lateral resolution of 2.3 \(\upmu\)m influenced discontinuities and showed shapes such as grains in the OCM images. As the shape of the cortex is curved three-dimensionally and the nerve fibers are distributed in the cortex, the OCM images were evaluated using Volume Viewer v.1.31 in Image J (v.1.49d) with different scales as shown in Figure 9. These orthoslice images were obtained with the threshold of 0 and resampling factor of 2 in ImageJ. The positions of the orthoslice images in Figure 9a,b are shown in Figure 8a with two dotted rectangles. As the resampling factor is 2 to reduce the noise, the 3D size of the area is the same, but the number of pixels is halved. The 3D size and pixels of Figure 9a are X 51.5 \(\upmu\)m 49 pixel, Y 51.5 \(\upmu\)m 49 pixel, Z 104 \(\upmu\)m 43 pixel and those in Figure 9b are X 14.9 \(\upmu\)m 13 pixel, Y 12.4 \(\upmu\)m 11 pixel, Z 8.1 \(\upmu\)m 4 pixel. (see film of Figure 9a,b)  
The short fibers and distributions in 3D space can be observed in Figure 9a and in the film files. Moreover, allowing for the differences of magnification, they are similar to the OCM images of an in vivo rodent cortex obtained with sealed windows as shown in Reference [36] (Figure 7c) and Reference [37] (Figure 6e). In the smaller 3D area, 3D shapes can also be observed in Figure 9b. The diameter of the imaged fibers is approximately determined as \(\sim\)2 \(\upmu\)m, comparable to the spatial resolutions. The practical diameters of the nerve fibers may be less than the spatial resolution; however, the rough 3D information such as connections and curves of imaged fibers can also be observed. However, owing to the reasons mentioned above, continuous and long 3D shapes of fibers have not been measured yet. Myelinated axons in the _en face_ plane were prominent in cortical layer I and IV-VI [35]. We investigated variations of the amounts of OCM image signals with changes in depth. At each IL, the dependence of sums of signal intensity in each _en face_ image on the scan depth up to 147 \(\upmu\)m for Rat 1 and Rat 2 at P4 are shown in Figure 10a,b, respectively. After subtracting the background, pixel values were added as the sum of signal intensities in the _en face_ image. These dependencies on the scan depth mainly depend on the depth of focus (DOF) in the OCM system and the distribution of backscatters such as nerve fibers. The DOF as the FWHM of contrast was obtained as 66 \(\upmu\)m as shown in Reference [30] (Figure 5). The sum of signal intensities decreased at a depth greater than approximately 80 \(\upmu\)m as shown in Figure 10a,b, owing to DOF. The dependence of these sums on depth is also due to local structures and distributions of scatterers in tissues. The sums of signal intensities are distributed in the depth direction from approximately 20 \(\upmu\)m to 90 \(\upmu\)m.  
Figure 9: (a) 3D orthoslice OCM image with the region of X 51.5 \(\upmu\)m, 49 pixel, Y 51.5 \(\upmu\)m, 49 pixel, Z 104 \(\upmu\)m, 43 pixel indicated by the dotted rectangle in Figure 8a (See Video S5); (b) 3D orthoslice OCM image with the region of X 14.9 \(\upmu\)m, 13 pixel, Y 12.4 \(\upmu\)m, 11 pixel, Z 8.1 \(\upmu\)m 4 pixel indicated by the smaller dotted rectangle in Figure 8a (Video S6). Assuming that neuron fibers dominate the _en face_ images owing to myelinated axons that are highly backscattering, the signal intensity increases with the amount of neuron fibers. For each IL, all the signal intensities within the measured volume (the FOV of _en face_ image by scanned depth) approximately indicate the amounts of nerve fibers. For Rat 1 and Rat 2 at positions P1 to P4, the dependences of the signal intensity integrations in the measured volume on the IL were obtained as shown in Figure 10c.