What is 3D Optical HREM imaging?

3D Optical HREM imaging is a technique that produces stacks of successive 2D tissue image samples, with a resolution almost equivalent to conventional histology. The technique delivers perfectly aligned images, that can be used to create superbly detailed 3D models.
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Optical imaging

A felixible, optical-based system, suitable for use over a broad range of magnifications that can be configured for multiple fluorescent channels.

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Resolution

Sections can be cut as thin as 1 μm, which results in 3D models with a typical voxel resolutions of 1-8 μm3. This resolution enables identification of blood vessels and nerves, often not visible using lower-resolution techniques such as optical projection tomography (OPT), micro-magnetic resonance imaging (μMRI) or micro-computed tomography (μCT).

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Applications

It is used in laboratories world-wide for 3D imaging of mouse embryos, embryonic organs, different tissue types and even plants.

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Speed

Samples can be imaged rapidly, as individual sections do not need to be collected, registered or stained. E.g. Sectioning an E14.5 mouse embryo at 3 microns would produce around 3,500 images in 8 hours.

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No sample distortion

Samples are embedded in a hard plastic resin that enables sections from 1 - 10 microns to be accurately removed.

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Alternate views

Image data produced can be used to calculate orthogonal and oblique views of the tissue sample, with little resolution lost. These views can provide important additional information about sample morphology.

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The technique

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Embedding

After preparation, samples are embedded in a plastic resin that has been made highly fluorescent by the addition of dyes, so opaque and translucent tissue is imaged by suppressing the fluorescence. This results in excellent quality, high resolution images of the block surface, irrespective of the tissue type or developmental stage - different tissue types are easily distinguishable.

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Sequential imaging

Once embedded, samples are sequentially imaged at a section thickness of 1-10 microns. Images are captured at the block surface, a section is removed, and the process repeated.

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Visualisation

Image data can subsequently be imported directly into 2D or 3D visualisation software for analysis.

Further information

  1. Pokhrel, N., E. Ben-Tal Cohen, O. Genin, D. Sela-Donenfeld and Y. Cinnamon
    Cellular and morphological characterization of blastoderms from freshly laid broiler eggs.
    Poult Sci, 2017 PubMed abstract
  2. Zhou, Z., J. Wang, C. Guo, W. Chang, J. Zhuang, P. Zhu, and X. Li
    Temporally Distinct Six2-Positive Second Heart Field Progenitors Regulate Mammalian Heart
    Development and Disease Cell Rep, 2017. 18(4), 1019-1032 PubMed abstract
  3. Le Garrec, J.F., J.N. Dominguez, A. Desgrange, K.D. Ivanovitch, E. Raphael, J.A. Bangham, M. Torres, E. Coen, T.J. Mohun, and S.M. Meilhac
    A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics.
    Elife, 2017. 6 PubMed abstract
  4. Geyer, S.H., L.F. Reissig, M. Husemann, C. Hofle, R. Wilson, F. Prin, D. Szumska, A. Galli, D.J. Adams, J. White, T.J. Mohun, and W.J. Weninger
    Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21-S23.
    J Anat, 2017. 231(4): 600-614 PubMed abstract
  5. Geyer, S.H., L. Reissig, J. Rose, R. Wilson, F. Prin, D. Szumska, R. Ramirez-Solis, C. Tudor, J. White, T.J. Mohun, and W.J. Weninger
    A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5). J Anat, 2017. 230(5): 710-719 PubMed abstract
  6. Geyer, S.H., B. Maurer-Gesek, L.F. Reissig and W.J. Weninger
    High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials.
    J Vis Exp, 2017(125) PubMed abstract
  7. Anderson, R.H., B. Jensen, T.J. Mohun, S.E. Petersen, N. Aung, F. Zemrak, R.N. Planken, and D.H. MacIver
    Key Questions Relating to Left Ventricular Noncompaction Cardiomyopathy: Is the Emperor Still Wearing Any Clothes?
    Can J Cardiol, 2017. 33(6): p. 747-757 PubMed abstract
  8. Aiello, V.D., D.E. Spicer, R.H. Anderson, N.A. Brown and T.J. Mohun
    The independence of the infundibular building blocks in the setting of double-outlet right ventricle.
    Cardiol Young, 2017. 27(5): p. 825-836 PubMed abstract
  9. Zak, J., V. Vives, D. Szumska, A. Vernet, J.E. Schneider, P. Miller, E.A. Slee, S. Joss, Y. Lacassie, E. Chen, L.F. Escobar, M. Tucker, A.S. Aylsworth, H.A. Dubbs, A.T. Collins, J. Andrieux, A. Dieux-Coeslier, E. Haberlandt, D. Kotzot, D.A. Scott, M.J. Parker, Z. Zakaria, Y.S. Choy, D. Wieczorek, A.M. Innes, K.R. Jun, S. Zinner, F. Prin, C.A. Lygate, P. Pretorius, J.A. Rosenfeld, T.J. Mohun, and X. Lu
    = ASPP2 deficiency causes features of 1q41q42 microdeletion syndrome.
    Cell Death Differ, 2016. 23(12): p. 1973-1984 PubMed abstract
  10. Wilson, R., S.H. Geyer, L. Reissig, J. Rose, D. Szumska, E. Hardman, F. Prin, C. McGuire, R. Ramirez-Solis, J. White, A. Galli, C. Tudor, E. Tuck, C. Mazzeo, J.C. Smith, E. Robertson, D.J. Adams, T. Mohun, and W.J. Weninger
    Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice. Wellcome Open Res, 2016. 1: p. 1 PubMed abstract
  11. 10. Lana-Elola, E., S. Watson-Scales, A. Slender, D. Gibbins, A. Martineau, C. Douglas, T. Mohun, E.M. Fisher, and V. Tybulewicz, Genetic dissection of Down syndrome- associated congenital heart defects using a new mouse mapping panel. Elife, 2016. 5. (https://www.ncbi.nlm.nih.gov/pubmed/26765563) 11. Huang, Y.C., F. Chen and X. Li, Clarification of mammalian cloacal morphogenesis using high-resolution episcopic microscopy. Dev Biol, 2016. 409(1): p. 106-13. (https://www.ncbi.nlm.nih.gov/pubmed/26485363) 12. Henkelman, R.M., M. Friedel, J.P. Lerch, R. Wilson and T. Mohun, Comparing homologous microscopic sections from multiple embryos using HREM. Dev Biol, 2016. 415(1): p. 1-5. (https://www.ncbi.nlm.nih.gov/pubmed/27208393) 13. Dickinson, M.E., A.M. Flenniken, X. Ji, L. Teboul, M.D. Wong, J.K. White, T.F. Meehan, W.J. Weninger, H. Westerberg, H. Adissu, C.N. Baker, L. Bower, J.M. Brown, L.B. Caddle, F. Chiani, D. Clary, J. Cleak, M.J. Daly, J.M. Denegre, B. Doe, M.E. Dolan, S.M. Edie, H. Fuchs, V. Gailus-Durner, A. Galli, A. Gambadoro, J. Gallegos, S. Guo, N.R. Horner, C.W. Hsu, S.J. Johnson, S. Kalaga, L.C. Keith, L. Lanoue, T.N. Lawson, M. Lek, M. Mark, S. Marschall, J. Mason, M.L. McElwee, S. Newbigging, L.M. Nutter, K.A. Peterson, R. Ramirez-Solis, D.J. Rowland, E. Ryder, K.E. Samocha, J.R. Seavitt, M. Selloum, Z. Szoke-Kovacs, M. Tamura, A.G. Trainor, I. Tudose, S. Wakana, J. Warren, O. Wendling, D.B. West, L. Wong, A. Yoshiki, C. International Mouse Phenotyping, L. Jackson, I.C.d.l.S. Infrastructure Nationale Phenomin, L. Charles River, M.R.C. Harwell, P. Toronto Centre for, I. Wellcome Trust Sanger, R.B. Center, D.G. MacArthur, G.P. Tocchini-Valentini, X. Gao, P. Flicek, A. Bradley, W.C. Skarnes, M.J. Justice, H.E. Parkinson, M. Moore, S. Wells, R.E. Braun, K.L. Svenson, M.H. de Angelis, Y. Herault, T. Mohun, A.M. Mallon, R.M. Henkelman, S.D. Brown, D.J. Adams, K.C. Lloyd, C. McKerlie, A.L. Beaudet, M. Bucan, and S.A. Murray, High-throughput discovery of novel developmental phenotypes. Nature, 2016. 537(7621): p. 508-514. (https://www.ncbi.nlm.nih.gov/pubmed/27626380) 14. Captur, G., R. Wilson, M.F. Bennett, G. Luxan, A. Nasis, J.L. de la Pompa, J.C. Moon, and T.J. Mohun, Morphogenesis of myocardial trabeculae in the mouse embryo. J Anat, 2016. 229(2): p. 314-25. (https://www.ncbi.nlm.nih.gov/pubmed/27020702) 15. Anderson, R.H., S. Mori, D.E. Spicer, N.A. Brown and T.J. Mohun, Development and Morphology of the Ventricular Outflow Tracts. World J Pediatr Congenit Heart Surg, 2016. 7(5): p. 561-77. (https://www.ncbi.nlm.nih.gov/pubmed/27587491) 16. Anderson, R.H., N.A. Brown and T.J. Mohun, Insights regarding the normal and abnormal formation of the atrial and ventricular septal structures. Clin Anat, 2016. 29(3): p. 290-304. (https://www.ncbi.nlm.nih.gov/pubmed/26378977) 17. Spicer, D.E., D.J. Henderson, B. Chaudhry, T.J. Mohun and R.H. Anderson, The anatomy and development of normal and abnormal coronary arteries. Cardiol Young, 2015. 25(8): p. 1493-503. (https://www.ncbi.nlm.nih.gov/pubmed/26675596) 18. Notari, M., Y. Hu, G. Sutendra, Z. Dedeic, M. Lu, L. Dupays, A. Yavari, C.A. Carr, S. Zhong, A. Opel, A. Tinker, K. Clarke, H. Watkins, D.J. Ferguson, D.P. Kelsell, S. de Noronha, M.N. Sheppard, M. Hollinshead, T.J. Mohun, and X. Lu, iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death. Proc Natl Acad Sci U S A, 2015. 112(9): p. E973-81. (https://www.ncbi.nlm.nih.gov/pubmed/25691752) 19. Matsui, H., S.Y. Ho, T.J. Mohun and H.M. Gardiner, Postmortem high-resolution episcopic microscopy (HREM) of small human fetal hearts. Ultrasound Obstet Gynecol, 2015. 45(4): p. 492-3. (https://www.ncbi.nlm.nih.gov/pubmed/25833371) 20. Geyer, S.H., I.E. Tinhofer, D.B. Lumenta, L.P. Kamolz, L. Branski, C.C. Finnerty, D.N. Herndon, and W.J. Weninger, High-resolution episcopic microscopy (HREM): a useful technique for research in wound care. Ann Anat, 2015. 197: p. 3-10. (https://www.ncbi.nlm.nih.gov/pubmed/25466930) 21. Dupays, L., C. Shang, R. Wilson, S. Kotecha, S. Wood, N. Towers, and T. Mohun, Sequential Binding of MEIS1 and NKX2-5 on the Popdc2 Gene: A Mechanism for Spatiotemporal Regulation of Enhancers during Cardiogenesis. Cell Rep, 2015. 13(1): p. 183-195. (https://www.ncbi.nlm.nih.gov/pubmed/26411676) 22. Bharucha, T., D.E. Spicer, T.J. Mohun, D. Black, G.W. Henry, and R.H. Anderson, Cor triatriatum or divided atriums: which approach provides the better understanding? Cardiol Young, 2015. 25(2): p. 193-207. (https://www.ncbi.nlm.nih.gov/pubmed/24820379) 23. Bailliard, F., D.E. Spicer, T.J. Mohun, G.W. Henry and R.H. Anderson, The problems that exist when considering the anatomic variability between the channels that permit interventricular shunting. Cardiol Young, 2015. 25(1): p. 15-28. (https://www.ncbi.nlm.nih.gov/pubmed/24865597) 24. Anderson, R.H., D.E. Spicer, G.W. Henry, C. Rigsby, A.M. Hlavacek, and T.J. Mohun, What is aortic overriding? Cardiol Young, 2015. 25(4): p. 612-25. (https://www.ncbi.nlm.nih.gov/pubmed/24983250) 25. Anderson, R.H., T.J. Mohun and N.A. Brown, Clarifying the morphology of the ostium primum defect. J Anat, 2015. 226(3): p. 244-57. (https://www.ncbi.nlm.nih.gov/pubmed/25676858) 26. Weninger, W.J., S.H. Geyer, A. Martineau, A. Galli, D.J. Adams, R. Wilson, and T.J. Mohun, Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy. Dis Model Mech, 2014. 7(10): p. 1143-52. (https://www.ncbi.nlm.nih.gov/pubmed/25256713) 27. Spicer, D.E., J.M. Bridgeman, N.A. Brown, T.J. Mohun and R.H. Anderson, The anatomy and development of the cardiac valves. Cardiol Young, 2014. 24(6): p. 1008-22. (https://www.ncbi.nlm.nih.gov/pubmed/25647375) 28. Rochais, F., R. Sturny, C.M. Chao, K. Mesbah, M. Bennett, T.J. Mohun, S. Bellusci, and R.G. Kelly, FGF10 promotes regional foetal cardiomyocyte proliferation and adult cardiomyocyte cell-cycle re-entry. Cardiovasc Res, 2014. 104(3): p. 432-42. (https://www.ncbi.nlm.nih.gov/pubmed/25344367) 29. Rana, M.S., M. Theveniau-Ruissy, C. De Bono, K. Mesbah, A. Francou, M. Rammah, J.N. Dominguez, M. Roux, B. Laforest, R.H. Anderson, T. Mohun, S. Zaffran, V.M. Christoffels, and R.G. Kelly, Tbx1 coordinates addition of posterior second heart field progenitor cells to the arterial and venous poles of the heart. Circ Res, 2014. 115(9): p. 790-9. (https://www.ncbi.nlm.nih.gov/pubmed/25190705) 30. Geyer, S.H., M.M. Nohammer, M. Matha, L. Reissig, I.E. Tinhofer, and W.J. Weninger, High-resolution episcopic microscopy (HREM): a tool for visualizing skin biopsies. Microsc Microanal, 2014. 20(5): p. 1356-64. (https://www.ncbi.nlm.nih.gov/pubmed/25198556) 31. Captur, G., L.R. Lopes, V. Patel, C. Li, P. Bassett, P. Syrris, D.M. Sado, V. Maestrini, T.J. Mohun, W.J. McKenna, V. Muthurangu, P.M. Elliott, and J.C. Moon, Abnormal cardiac formation in hypertrophic cardiomyopathy: fractal analysis of trabeculae and preclinical gene expression. Circ Cardiovasc Genet, 2014. 7(3): p. 241-8. (https://www.ncbi.nlm.nih.gov/pubmed/24704860) 32. Anderson, R.H., D.E. Spicer, N.A. Brown and T.J. Mohun, The development of septation in the four-chambered heart. Anat Rec (Hoboken), 2014. 297(8): p. 1414-29. (https://www.ncbi.nlm.nih.gov/pubmed/24863187) 33. Vettukattil, J.J., Z. Ahmed, A.P. Salmon, T. Mohun and R.H. Anderson, Defects in the oval fossa: morphologic variations and impact on transcatheter closure. J Am Soc Echocardiogr, 2013. 26(2): p. 192-9. (https://www.ncbi.nlm.nih.gov/pubmed/23265438) 34. Norris, F.C., M.D. Wong, N.D. Greene, P.J. Scambler, T. Weaver, W.J. Weninger, T.J. Mohun, R.M. Henkelman, and M.F. Lythgoe, A coming of age: advanced imaging technologies for characterising the developing mouse. Trends Genet, 2013. 29(12): p. 700-11. (https://www.ncbi.nlm.nih.gov/pubmed/24035368) 35. Maurer, B., S.H. Geyer and W.J. Weninger, A chick embryo with a yet unclassified type of cephalothoracopagus malformation and a hypothesis for explaining its genesis. Anat Histol Embryol, 2013. 42(3): p. 191-200. (https://www.ncbi.nlm.nih.gov/pubmed/22971166) 36. Captur, G., V. Muthurangu, C. Cook, A.S. Flett, R. Wilson, A. Barison, D.M. Sado, S. Anderson, W.J. McKenna, T.J. Mohun, P.M. Elliott, and J.C. Moon, Quantification of left ventricular trabeculae using fractal analysis. J Cardiovasc Magn Reson, 2013. 15: p. (https://www.ncbi.nlm.nih.gov/pubmed/23663522) 37. Breckenridge, R.A., I. Piotrowska, K.E. Ng, T.J. Ragan, J.A. West, S. Kotecha, N. Towers, M. Bennett, P.C. Kienesberger, R.T. Smolenski, H.K. Siddall, J.L. Offer, M.M. Mocanu, D.M. Yelon, J.R. Dyck, J.L. Griffin, A.Y. Abramov, A.P. Gould, and T.J. Mohun, Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism. PLoS Biol, 2013. 11(9): p. e1001666. (https://www.ncbi.nlm.nih.gov/pubmed/24086110) 38. Bamforth, S.D., B. Chaudhry, M. Bennett, R. Wilson, T.J. Mohun, L.H. Van Mierop, D.J. Henderson, and R.H. Anderson, Clarification of the identity of the mammalian fifth pharyngeal arch artery. Clin Anat, 2013. 26(2): p. 173-82. (https://www.ncbi.nlm.nih.gov/pubmed/22623372) 39. Anderson, R.H., D.E. Spicer, J.M. Giroud and T.J. Mohun, Tetralogy of Fallot: nosological, morphological, and morphogenetic considerations. Cardiol Young, 2013. 23(6): p. 858-66. (https://www.ncbi.nlm.nih.gov/pubmed/24401259) 40. Anderson, R.H., N.A. Brown, T.J. Mohun and A.F. Moorman, Insights from cardiac development relevant to congenital defects and adult clinical anatomy. J Cardiovasc Transl Res, 2013. 6(2): p. 107-17. (https://www.ncbi.nlm.nih.gov/pubmed/23225336) 41. Sizarov, A., W.H. Lamers, T.J. Mohun, N.A. Brown, R.H. Anderson, and A.F. Moorman, Three-dimensional and molecular analysis of the arterial pole of the developing human heart. J Anat, 2012. 220(4): p. 336-49. (https://www.ncbi.nlm.nih.gov/pubmed/22296102) 42. Mohun, T.J. and W.J. Weninger, Episcopic three-dimensional imaging of embryos. Cold Spring Harb Protoc, 2012. 2012(6): p. 641-6. (https://www.ncbi.nlm.nih.gov/pubmed/22661435) 43. Mohun, T.J. and W.J. Weninger, Embedding embryos for episcopic fluorescence image capturing (EFIC). Cold Spring Harb Protoc, 2012. 2012(6): p. 675-7. (https://www.ncbi.nlm.nih.gov/pubmed/22661436) 44. Mohun, T.J. and W.J. Weninger, Embedding embryos for high-resolution episcopic microscopy (HREM). Cold Spring Harb Protoc, 2012. 2012(6): p. 678-80. (https://www.ncbi.nlm.nih.gov/pubmed/22661437) 45. Mohun, T.J. and W.J. Weninger, Generation of volume data by episcopic three- dimensional imaging of embryos. Cold Spring Harb Protoc, 2012. 2012(6): p. 681-2. (https://www.ncbi.nlm.nih.gov/pubmed/22661438) 46. Lescroart, F., T. Mohun, S.M. Meilhac, M. Bennett and M. Buckingham, Lineage tree for the venous pole of the heart: clonal analysis clarifies controversial genealogy based on genetic tracing. Circ Res, 2012. 111(10): p. 1313-22. (https://www.ncbi.nlm.nih.gov/pubmed/22855565) 47. Gindes, L., H. Matsui, R. Achiron, T. Mohun, S.Y. Ho, and H. Gardiner, Comparison of ex-vivo high-resolution episcopic microscopy with in-vivo four-dimensional high- resolution transvaginal sonography of the first-trimester fetal heart. Ultrasound Obstet Gynecol, 2012. 39(2): p. 196-202. (https://www.ncbi.nlm.nih.gov/pubmed/21638368) 48. Anderson, R.H., B. Chaudhry, T.J. Mohun, S.D. Bamforth, D. Hoyland, H.M. Phillips, S. Webb, A.F. Moorman, N.A. Brown, and D.J. Henderson, Normal and abnormal development of the intrapericardial arterial trunks in humans and mice. Cardiovasc Res, 2012. 95(1): p. 108-15. (https://www.ncbi.nlm.nih.gov/pubmed/22499773) 49. Mohun, T.J. and W.J. Weninger, Imaging heart development using high-resolution episcopic microscopy. Curr Opin Genet Dev, 2011. 21(5): p. 573-8. (https://www.ncbi.nlm.nih.gov/pubmed/21893408) 50. de Boer, B.A., A.T. Soufan, J. Hagoort, T.J. Mohun, M.J. van den Hoff, A. Hasman, F.P. Voorbraak, A.F. Moorman, and J.M. Ruijter, The interactive presentation of 3D information obtained from reconstructed datasets and 3D placement of single histological sections with the 3D portable document format. Development, 2011. 138(1): p. 159-67. (https://www.ncbi.nlm.nih.gov/pubmed/21138978) 51. Anderson, R.H., T.J. Mohun and A.F. Moorman, What is a ventricle? Cardiol Young, 2011. 21 Suppl 2: p. 14-22. (https://www.ncbi.nlm.nih.gov/pubmed/22152524) 52. Matsui, H., T. Mohun and H.M. Gardiner, Three-dimensional reconstruction imaging of the human foetal heart in the first trimester. Eur Heart J, 2010. 31(4): p. 415. (https://www.ncbi.nlm.nih.gov/pubmed/19965862) 53. Dunlevy, L., M. Bennett, A. Slender, E. Lana-Elola, V.L. Tybulewicz, E.M. Fisher, and T. Mohun, Down's syndrome-like cardiac developmental defects in embryos of the transchromosomic Tc1 mouse. Cardiovasc Res, 2010. 88(2): p. 287-95. (https://www.ncbi.nlm.nih.gov/pubmed/20558441) 54. Anderson, R.H., A. Cook, N.A. Brown, D.J. Henderson, B. Chaudhry, and T. Mohun, Development of the outflow tracts with reference to aortopulmonary windows and aortoventricular tunnels. Cardiol Young, 2010. 20 Suppl 3: p. 92-9. (https://www.ncbi.nlm.nih.gov/pubmed/21087564) 55. Geyer, S.H., T.J. Mohun and W.J. Weninger, Visualizing vertebrate embryos with episcopic 3D imaging techniques. ScientificWorldJournal, 2009. 9: p. 1423-37. (https://www.ncbi.nlm.nih.gov/pubmed/20024516) 56. Weninger, W.J. and T.J. Mohun, Three-dimensional analysis of molecular signals with episcopic imaging techniques. Methods Mol Biol, 2007. 411: p. 35-46. (https://www.ncbi.nlm.nih.gov/pubmed/18287637) 57. Weninger, W.J., S.H. Geyer, T.J. Mohun, D. Rasskin-Gutman, T. Matsui, I. Ribeiro, F. Costa Lda, J.C. Izpisua-Belmonte, and G.B. Muller, High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology. Anat Embryol (Berl), 2006. 211(3): p. 213-21. (https://www.ncbi.nlm.nih.gov/pubmed/16429276) 58. Going, J.J. and T.J. Mohun, Human breast duct anatomy, the 'sick lobe' hypothesis and intraductal approaches to breast cancer. Breast Cancer Res Treat, 2006. 97(3): p. 285-91. (https://www.ncbi.nlm.nih.gov/pubmed/16791485)

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