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Peer-reviewedCC BY 4.0Frontiers in Neuroscience

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

  • Danny F. Xie1
  • Christian Crouzet1
  • Wei Ling Lau2
  • David H. Cribbs3
  • Mark J. Fisher1,4,5
  • Bernard Choi1,6,7
7 affiliations
  1. 1Beckman Laser Institute and Medical Clinic, University of California, Irvine
  2. 2Department of Medicine, Division of Nephrology, University of California, Irvine
  3. 3Institute for Memory Impairments and Neurological Disorders, University of California, Irvine
  4. 4Department of Neurology, University of California, Irvine
  5. 5Department of Pathology & Laboratory Medicine, University of California, Irvine
  6. 6Department of Biomedical Engineering, University of California, Irvine
  7. 7Department of Surgery, University of California, Irvine
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Abstract

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.

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Cite

BibTeXRIS
BibTeX
@article{xie2026confocal,
  title = {{3D confocal imaging reveals quantitative bias in cerebral microhemorrhage counts from standard histology}},
  author = {Danny F. Xie and Christian Crouzet and Wei Ling Lau and David H. Cribbs and Mark J. Fisher and Bernard Choi},
  journal = {Frontiers in Neuroscience},
  year = {2026},
  month = oct,
  doi = {10.3389/fnins.2026.1940336},
  url = {https://www.frontiersin.org/articles/10.3389/fnins.2026.1940336/full},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - 3D confocal imaging reveals quantitative bias in cerebral microhemorrhage counts from standard histology
AU  - Danny F. Xie
AU  - Christian Crouzet
AU  - Wei Ling Lau
AU  - David H. Cribbs
AU  - Mark J. Fisher
AU  - Bernard Choi
PY  - 2026
DA  - 2026/10/27
JO  - Frontiers in Neuroscience
DO  - 10.3389/fnins.2026.1940336
UR  - https://www.frontiersin.org/articles/10.3389/fnins.2026.1940336/full
AB  - 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.
SN  - 1662-453X
ER  -