Fig. 5. Stereological evaluation on LSFM reconstructions.

(A) A slab from the LSFM imaging dataset stained with the anti-NeuN antibody, displaying a systematic-random counting grid used during stereologic analyses. The grid is placed over the infragranular layers (yellow outline), and optical disector frames are placed in all segments of the grid that are located within the region of interest. (B) Different z-levels within a disector with color markers indicating NeuN⁺ neurons that have been sampled, according to stereologic rules (see Materials and Methods), at different focal planes in the disector thickness (24). The neurons are marked, from left to right, in blue, purple, yellow, and red as they appeared in focus moving up in the z axis. Neurons that touch or cross the inclusion (green) sides of the disector are included in the sampling at this site, whereas those touching or crossing the exclusion (red) sides of the disector are not, giving each NeuN⁺ neuron in this region of interest the same probability to be counted only once. (C and D) The registration of stereologic data onto the LSFM dataset for the entire slab. Each red dot in (C) corresponds to NeuN⁺ neurons sampled through the slab within an optical disector depth during stereologic analysis and used to generate an estimate of the total population number. The green dots in (D) represent CR⁺ neurons that were analyzed only in layers 2 and 3 as they are very sparse in the deep layers of the neocortex. Grid size in (A) = 800 μm, and counting frame in (B) = 150 μm.