3D confocal imaging reveals quantitative bias in cerebral microhemorrhage counts from standard histology

Standard histological quantification of cerebral microhemorrhages (CMH), the pathologic substrate of cerebral microbleeds, may systematically undercount lesion burden due to sparse tissue sampling. CMH are visualized using Prussian blue-stained 2D tissue sections. This method provides a snapshot of CMH lesions within a brain but does not capture comprehensive quantification across the entire brain. Tissue clearing combined with confocal microscopy can visualize intact CMH lesions in large tissue samples without tissue sectioning. We collected three-dimensional confocal images of optically-cleared, 1-mm-thick brain sections. We compared CMH counts from comprehensive three-dimensional imaging to those obtained from simulated standard two-dimensional histological sectioning. Sparse sampling (one 40-μm-thick section out of every six) resulted in the omission of 38% of individual CMH, disproportionately affecting smaller lesions. Increasing sectioning frequency to one in every 2–3 sections lowered the miss rate to 13%–20% but concurrently increased the likelihood of counting single lesions multiple times. These results demonstrate that tissue sampling frequency substantially affects CMH quantification. Our data suggest using 40-μm sections at a 1-every-4 frequency to provide a balance between omission of CMH, repeat counting of CMH, and workload. This recommendation may change for different study designs and/or experimental models.