Thicker L2/L3 in human cortex is accompanied by lower neuronal densities and larger cell body area in layer 3. (a) Microstructure analysis workflow: in NeuN stained human cortical slice multiple ROIs were selected for analysis; within each ROI a region of interest was defined that included only L2/L3 and was divided in four sublayers of equal thickness (each at 25% of total L2/L3 thickness). The neurons were detected from the images using custom-made image-processing scripts (detected neurons are shown in red). (b) Examples of NeuN stained slices from two subjects showing different L2/L3 thickness. (c) Neuronal density decreases, and cell body area increases from L2 to deeper sublayers of L3 (black circles are mean data from 16 subjects; 24 slices, 113 ROIs). (d) Results of neuronal density correlation to L2/L3 thickness per sublayer: neuronal density correlates stronger to L2/L3 thickness in deeper layer 3: correlation coefficients (R) and variance explained (R²) are shown per sublayer, P-values are color coded (^*P-value<0.05; ^**P-value<0.01). (e) Thicker L2/3 shows negative association with neuronal density in L3 (F(1,14) = 9.15) and positive association with cell body area (F(1,14) = 5.88). black circles are means per subject, n = 16, gray circles are ROIs, n = 113, black lines are linear regression fits to subject level data, shaded area (blue) represents 95% confidence bounds.