6.9 Conclusions
Cryoembedding in Lowicryl HM-20 preserves both antigenicity and ultrastructure
of brain tissue, enabling conjugate array tomography. Using fluorescence microscopy, dozens of different antibodies and other fluorescent markers can be imaged
at synaptic resolution within large volumes of tissue. The same arrays can also be
viewed using field emission SEM, which allows for the classic benefits of electron
microscopy. Such integration of different imaging modalities can substantially
enrich our understanding of the brain.
References
1. J. Roth, The silver anniversary of gold: 25 years of the colloidal gold marker system for
immunocytochemistry and histochemistry. Histochem. Cell Biol. 106(1), 1–8 (1996)
2. B.L. Wang, L.I. Larsson, Simultaneous demonstration of multiple antigens by indirect
immunofluorescence or immunogold staining. Novel light and electron microscopical double
and triple staining method employing primary antibodies from the same species.
Histochemistry 83(1), 47–56 (1985)
3. D.A. Meyer, J.A. Oliver, R.M. Albrecht, Colloidal palladium particles of different shapes for
electron microscopy labeling. Microsc. Microanal. 16(1), 33–42 (2010)
4. B.N.G. Giepmans, T.J. Deerinck, B.L. Smarr, Y.Z. Jones, M.H. Ellisman, Correlated light and
electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat.
Methods 2, 743–749 (2005)
5. D.R. Glenn, H. Zhang, N. Kasthuri, R. Schalek, P.K. Lo, A.S. Trifonov, H. Park, J.W.
Lichtman, R.L. Walsworth, Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours. Sci. Rep. 2, 865 (2012)
6. W. Schubert, B. Bonnekoh, A.J. Pommer, L. Philipsen, R. Böckelmann, Y. Malykh, H.
Gollnick, M. Friedenberger, M. Bode, A.W. Dress, Analyzing proteome topology and
function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24(10),
1270–1278 (2006)
7. M. Bode, M. Irmler, M. Friedenberger, C. May, K. Jung, C. Stephan, H.E. Meyer, C. Lach, R.
Hillert, A. Krusche, J. Beckers, K. Marcus, W. Schubert, Interlocking transcriptomics,
proteomics and toponomics technologies for brain tissue analysis in murine hippocampus.
Proteomics 8(6), 1170–1178 (2008)
8. W. Schubert, Systematic, spatial imaging of large multimolecular assemblies and the
emerging principles of supramolecular order in biological systems. J. Mol. Recognit. 27(1),
3–18 (2014)
9. K.D. Micheva, S.J. Smith, Array tomography: a new tool for imaging the molecular
architecture and ultrastructure of neural circuits. Neuron 55(1), 25–36 (2007)
10. K.D. Micheva, B. Busse, N.C. Weiler, N. O’Rourke, S.J. Smith, Single-synapse analysis of a
diverse synapse population: proteomic imaging methods and markers. Neuron 68(4), 639–653
(2010)
11. G. Wang, S.J. Smith, Sub-diffraction limit localization of proteins in volumetric space using
Bayesian restoration of fluorescence images from ultrathin specimens. PLoS Comput. Biol. 8
(8), e1002671 (2012)
12. A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang, Superresolution imaging of chemical
synapses in the brain. Neuron 68(5), 843–856 (2010)
6 Conjugate Immunofluorescence—SEM Array Tomography …
179
Cryoembedding in Lowicryl HM-20 preserves both antigenicity and ultrastructure
of brain tissue, enabling conjugate array tomography. Using fluorescence microscopy, dozens of different antibodies and other fluorescent markers can be imaged
at synaptic resolution within large volumes of tissue. The same arrays can also be
viewed using field emission SEM, which allows for the classic benefits of electron
microscopy. Such integration of different imaging modalities can substantially
enrich our understanding of the brain.
References
1. J. Roth, The silver anniversary of gold: 25 years of the colloidal gold marker system for
immunocytochemistry and histochemistry. Histochem. Cell Biol. 106(1), 1–8 (1996)
2. B.L. Wang, L.I. Larsson, Simultaneous demonstration of multiple antigens by indirect
immunofluorescence or immunogold staining. Novel light and electron microscopical double
and triple staining method employing primary antibodies from the same species.
Histochemistry 83(1), 47–56 (1985)
3. D.A. Meyer, J.A. Oliver, R.M. Albrecht, Colloidal palladium particles of different shapes for
electron microscopy labeling. Microsc. Microanal. 16(1), 33–42 (2010)
4. B.N.G. Giepmans, T.J. Deerinck, B.L. Smarr, Y.Z. Jones, M.H. Ellisman, Correlated light and
electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat.
Methods 2, 743–749 (2005)
5. D.R. Glenn, H. Zhang, N. Kasthuri, R. Schalek, P.K. Lo, A.S. Trifonov, H. Park, J.W.
Lichtman, R.L. Walsworth, Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours. Sci. Rep. 2, 865 (2012)
6. W. Schubert, B. Bonnekoh, A.J. Pommer, L. Philipsen, R. Böckelmann, Y. Malykh, H.
Gollnick, M. Friedenberger, M. Bode, A.W. Dress, Analyzing proteome topology and
function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24(10),
1270–1278 (2006)
7. M. Bode, M. Irmler, M. Friedenberger, C. May, K. Jung, C. Stephan, H.E. Meyer, C. Lach, R.
Hillert, A. Krusche, J. Beckers, K. Marcus, W. Schubert, Interlocking transcriptomics,
proteomics and toponomics technologies for brain tissue analysis in murine hippocampus.
Proteomics 8(6), 1170–1178 (2008)
8. W. Schubert, Systematic, spatial imaging of large multimolecular assemblies and the
emerging principles of supramolecular order in biological systems. J. Mol. Recognit. 27(1),
3–18 (2014)
9. K.D. Micheva, S.J. Smith, Array tomography: a new tool for imaging the molecular
architecture and ultrastructure of neural circuits. Neuron 55(1), 25–36 (2007)
10. K.D. Micheva, B. Busse, N.C. Weiler, N. O’Rourke, S.J. Smith, Single-synapse analysis of a
diverse synapse population: proteomic imaging methods and markers. Neuron 68(4), 639–653
(2010)
11. G. Wang, S.J. Smith, Sub-diffraction limit localization of proteins in volumetric space using
Bayesian restoration of fluorescence images from ultrathin specimens. PLoS Comput. Biol. 8
(8), e1002671 (2012)
12. A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang, Superresolution imaging of chemical
synapses in the brain. Neuron 68(5), 843–856 (2010)
6 Conjugate Immunofluorescence—SEM Array Tomography …
179