Figure 1: Minicolumnar structure in mouse V1 and three-dimensional imaging.

(a) Nissl-stained fixed section of the mouse V1. Vertical arrays of cells are visible that span cortical layers. Scale bars, 100 μm. (b) A 2D (x–z) colour-coded probability density map of neighbouring cell location in Nissl-stained image in a. (c) 1D projection plot of b. The 2D map in b is averaged along z-axis in the range of ±35 μm. Significant regions (b) and peaks (c) are indicated by black lines (FDR adjusted P<0.05). (d) Uncorrected P value map corresponding the probability density map (b). P values are computed by a randomization procedure (see Methods) and indicated by colour code. White area indicates P≥0.05. (e) A high-speed three-dimensional two-photon microscope used in the present study. The combination of a resonant (x-axis) and a galvano scanner (y-axis) and a piezo z-axis drive enables rapid three-dimensional imaging. (f) An x–z section from a OGB-1-stained volume obtained in vivo using two-photon microscopy, showing vertical alignment of cells. The red dotted line shows the axial inclination of the volume. Scale bars, 30 μm. (g) A 3D probability density map of neighbouring cells sectioned at x–z plane. The peaks along the z-axis show a vertical alignment of neighbouring cells in the same minicolumns. The angle of inclination of the volume from the z-axis (green line) is calculated (red dotted line, 2° in this case) , and the volume image is corrected by tilting. Scale bars, 10 μm. (h) A 2D colour-coded probability density map of neighbouring cells in layer 2/3. Several hot spots can be observed, and are regularly spaced at ∼15 μm. The radius of the centre peak is 5.1 μm by fitting with a 2D Gaussian function. Significant areas are contoured by grey lines (FDR adjusted P<0.05). (i) Uncorrected P value map corresponding to the probability density map in h . White area indicates P≥0.05.