Anatomy and Embryology

, Volume 211, Issue 3, pp 213–221 | Cite as

High-resolution episcopic microscopy: a rapid technique for high detailed 3D analysis of gene activity in the context of tissue architecture and morphology

  • Wolfgang J. Weninger
  • Stefan H. Geyer
  • Timothy J. Mohun
  • Diego Rasskin-Gutman
  • Takaaki Matsui
  • Ines Ribeiro
  • Luciano da F. Costa
  • Juan Carlos Izpisúa-Belmonte
  • Gerd B. Müller
Original Article

Abstract

We describe a new methodology for rapid 2D and 3D computer analysis and visualisation of gene expression and gene product pattern in the context of anatomy and tissue architecture. It is based on episcopic imaging of embryos and tissue samples, as they are physically sectioned, thereby producing inherently aligned digital image series and volume data sets, which immediately permit the generation of 3D computer representations. The technique uses resin as embedding medium, eosin for unspecific tissue staining, and colour reactions (β-galactosidase/Xgal or BCIP/NBT) for specific labelling of gene activity and mRNA pattern. We tested the potential of the method for producing high-resolution volume data sets of adult human and porcine tissue samples and of specifically and unspecifically stained mouse, chick, quail, frog, and zebrafish embryos. The quality of the episcopic images resembles the quality of digital images of true histological sections with respect to resolution and contrast. Specifically labelled structures can be extracted using simple thresholding algorithms. Thus, the method is capable of quickly and precisely detecting molecular signals simultaneously with anatomical details and tissue architecture. It has no tissue restrictions and can be applied for analysis of human tissue samples as well as for analysis of all developmental stages of embryos of a wide variety of biomedically relevant species.

Keywords

3D reconstruction Gene expression analysis Anatomy Embryology RNA pattern 

References

  1. Blasberg RG, Tjuvajev JG (2003) Molecular-genetic imaging: current and future perspectives. J Clin Invest 111(11):1620–1629CrossRefPubMedGoogle Scholar
  2. Costa LdaF, Barbosa MS, Manoel ET, Streicher J, Müller GB (2004) Mathematical characterization of three-dimensional gene expression patterns. Bioinformatics 20(11):1653–1662CrossRefPubMedGoogle Scholar
  3. Costa LdaF, Beletti ME, Müller GB, Rasskin-Gutman D, Sternik G, Ibanes M, Belmonte JCI (2005) Field approach to three-dimensional gene expression pattern characterization. Appl Phys Lett 86(14):143901CrossRefGoogle Scholar
  4. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11):e329CrossRefPubMedGoogle Scholar
  5. Ewald AJ, McBride H, Reddington M, Fraser SE, Kerschmann R (2002) Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution. Dev Dyn 225(3):369–375CrossRefPubMedGoogle Scholar
  6. Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195(4):231–272PubMedGoogle Scholar
  7. Herschman HR (2003) Molecular imaging: looking at problems, seeing solutions. Science 302(5645):605–608CrossRefPubMedGoogle Scholar
  8. Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305(5686):1007–1009CrossRefPubMedGoogle Scholar
  9. Jacobs RE, Fraser SE (1994) Magnetic resonance microscopy of embryonic cell lineages and movements. Science 263(5147):681–684PubMedCrossRefGoogle Scholar
  10. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310PubMedGoogle Scholar
  11. Kolker SJ, Tajchman U, Weeks DL (2000) Confocal imaging of early heart development in Xenopus laevis. Dev Biol 218(1):64–73CrossRefPubMedGoogle Scholar
  12. Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Fraser SE, Meade TJ (2000) In vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 18(3):321–325CrossRefPubMedGoogle Scholar
  13. Massoud TF, Gambhir SS (2003) Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17(5):545–580CrossRefPubMedGoogle Scholar
  14. Matsui T, Raya A, Kawakami Y, Callol-Massot C, Capdevila J, Rodriguez-Esteban C, Izpisua Belmonte JC (2005) Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development. Genes Dev 19(1):164–175CrossRefPubMedGoogle Scholar
  15. Ng JK, Kawakami Y, Buscher D, Raya A, Itoh T, Koth CM, Rodriguez Esteban C, Rodriguez-Leon J, Garrity DM, Fishman MC, Izpisua Belmonte JC (2002) The limb identity gene Tbx5 promotes limb initiation by interacting with Wnt2b and Fgf10. Development 129(22):5161–5170PubMedGoogle Scholar
  16. Schneider JE, Bamforth SD, Farthing CR, Clarke K, Neubauer S, Bhattacharya S (2003) High-resolution imaging of normal anatomy, and neural and adrenal malformations in mouse embryos using magnetic resonance microscopy. J Anat 202(2):239–247CrossRefPubMedGoogle Scholar
  17. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sørensen J, Baldock R, Davidson D (2002) Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296(5567):541–545CrossRefPubMedGoogle Scholar
  18. Streicher J, Weninger WJ, Müller GB (1997) External marker-based automatic congruencing: a new method of 3D reconstruction from serial sections. Anat Rec 248(4):583–602CrossRefPubMedGoogle Scholar
  19. Streicher J, Donat MA, Strauss B, Sporle R, Schughart K, Müller GB (2000) Computer-based three-dimensional visualization of developmental gene expression. Nat Genet 25(2):147–152CrossRefPubMedGoogle Scholar
  20. Weissleder R, Ntziachristos V (2003) Shedding light onto live molecular targets. Nat Med 9(1):123–128CrossRefPubMedGoogle Scholar
  21. Weninger WJ, Mohun T (2002) Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing. Nat Genet 30(1):59–65CrossRefPubMedGoogle Scholar
  22. Weninger WJ, Meng S, Streicher J, Müller GB (1998) A new episcopic method for rapid 3-D reconstruction: applications in anatomy and embryology. Anat Embryol (Berl) 197(5):341–348CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Wolfgang J. Weninger
    • 1
  • Stefan H. Geyer
    • 1
  • Timothy J. Mohun
    • 2
  • Diego Rasskin-Gutman
    • 3
  • Takaaki Matsui
    • 4
  • Ines Ribeiro
    • 3
  • Luciano da F. Costa
    • 5
  • Juan Carlos Izpisúa-Belmonte
    • 3
  • Gerd B. Müller
    • 6
  1. 1.Integrative Morphology Group, Center for Anatomy & Cell BiologyMedical University of ViennaViennaAustria
  2. 2.Developmental Biology DivisionNational Institute for Medical ResearchLondonUK
  3. 3.GELThe Salk InstituteLa JollaUSA
  4. 4.Nara Institute of Science and TechnologyTakayamaJapan
  5. 5.Institute of Physics at São CarlosUniversity of São PauloSão PauloBrazil
  6. 6.Department of Theoretical BiologyUniversity of ViennaViennaAustria

Personalised recommendations