Applied Microscopy

  • Volume 50
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  • Pages.14.1-14.6
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  • 2020
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  • 2287-5123(pISSN)
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  • 2287-4445(eISSN)
Korean Society of Microscopy (한국현미경학회)
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Double staining method for array tomography using scanning electron microscopy

  • Eunjin Kim (National Instrumentation Center for Environmental Management, Seoul National University) ;
  • Jiyoung Lee (National Instrumentation Center for Environmental Management, Seoul National University) ;
  • Seulgi Noh (Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science & Technology (DGIST)) ;
  • Ohkyung Kwon (National Instrumentation Center for Environmental Management, Seoul National University) ;
  • Ji Young Mun (Neural circuit research group, Korea Brain Research Institute)
  • Received : 2020.04.02
  • Accepted : 2020.06.05
  • Published : 2020.12.31
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Abstract

Scanning electron microscopy (SEM) plays a central role in analyzing structures by imaging a large area of brain tissue at nanometer scales. A vast amount of data in the large area are required to study structural changes of cellular organelles in a specific cell, such as neurons, astrocytes, oligodendrocytes, and microglia among brain tissue, at sufficient resolution. Array tomography is a useful method for large-area imaging, and the osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods are commonly used to enhance membrane contrast. Because many samples prepared using the conventional technique without en bloc staining are considered inadequate for array tomography, we suggested an alternative technique using post-staining conventional samples and compared the advantages.

Keywords

Acknowledgement

The instruments (scanning electron microscopy) were supplied by the Brain Research Core Facilities at KBRI and NICEM.

References

  1. V. Baena, R.L. Schalek, J.W. Lichtman, M. Terasaki, Serial-section electron microscopy using automated tape-collecting ultramicrotome (ATUM). Methods Cell Biol. 152, 41-67 (2019). https://doi.org/10.1016/bs.mcb.2019.04.004
  2. C. Bosch, A. Martinez, N. Masachs, C.M. Teixeira, I. Fernaud, F. Ulloa, E. Perez-Martinez, C. Lois, J.X. Comella, J. DeFelipe, A. Merchan-Perez, E. Soriano, FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons. Front. Neuroanat. 9, 60 (2015). https://doi.org/10.3389/fnana.2015.00060
  3. K.L. Briggman, D.D. Bock, Volume electron microscopy for neuronal circuit reconstruction. Curr. Opin. Neurobiol. 22(1), 154-161 (2012). https://doi.org/10.1016/j.conb.2011.10.022
  4. W. Denk, H. Horstmann, Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2(11), e329 (2004). https://doi.org/10.1371/journal.pbio.0020329
  5. D.H. Hall, E. Hartwieg, K.C. Nguyen, Modern electron microscopy methods for C. elegans. Methods Cell Biol. 107, 93-149 (2012). https://doi.org/10.1016/B978-0-12-394620-1.00004-7
  6. K.J. Hayworth, J.L. Morgan, R. Schalek, D.R. Berger, D.G. Hildebrand, J.W. Lichtman, Imaging ATUM ultrathin section libraries with WaferMapper: A multi-scale approach to EM reconstruction of neural circuits. Front. Neural Circuits. 8, 68 (2014). https://doi.org/10.3389/fncir.2014.00068
  7. Y. Hua, P. Laserstein, M. Helmstaedter, Large-volume en bloc staining for electron microscopy-based connectomics. Nat. Commun. 6, 7923 (2015). https://doi.org/10.1038/ncomms8923
  8. Y. Kubota, J. Sohn, S. Hatada, M. Schurr, J. Straehle, A. Gour, R. Neujahr, T. Miki, S. Mikula, Y. Kawaguchi, A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure. Nat. Commun. 9(1), 437 (2018a). https://doi.org/10.1038/s41467-017-02768-7
  9. Y. Kubota, J. Sohn, Y. Kawaguchi, Large volume electron microscopy and neural microcircuit analysis. Front. Neural Circuits 12, 98 (2018b). https://doi.org/10.3389/fncir.2018.00098
  10. S. Lippens, A. Kremer, P. Borghgraef, C.J. Guerin, Serial block face-scanning electron microscopy for volume electron microscopy. Methods Cell Biol. 152, 69-85 (2019). https://doi.org/10.1016/bs.mcb.2019.04.002
  11. 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, 639-653 (2010) https://doi.org/10.1016/j.neuron.2010.09.024
  12. D. Oberti, M.A. Kirschmann, R.H. Hahnloser, Projection neuron circuits resolved using correlative array tomography. Front Neurosci 5, 50 (2011) https://doi.org/10.3389/fnins.2011.0005
  13. A.M. Seligman, H.L. Wasserkrug, J.S. Hanker, A new staining method (OTO) for enhancing contrast of lipid--containing membranes and droplets in osmium tetroxide--fixed tissue with osmiophilic thiocarbohydrazide (TCH). J. Cell Biol. 30(2), 424-432 (1966). https://doi.org/10.1083/jcb.30.2.424
  14. A.M. Steyer, A. Schertel, C. Nardis, W. Mobius, FIB-SEM of mouse nervous tissue: Fast and slow sample preparation. Methods Cell Biol. 152, 1-21 (2019). https://doi.org/10.1016/bs.mcb.2019.03.009
  15. L. de Vivo, M. Bellesi, W. Marshall, E.A. Bushong, M.H. Ellisman, G. Tononi, Cirelli C. Ultrastructural evidence for synaptic scaling across the wake/sleep cycle. Science. 355(6324), 507-510 (2017). https://doi.org/10.1126/science.aah5982
  16. A.A. Wanner, M.A. Kirschmann, C. Genoud, Challenges of microtome-based serial block-face scanning electron microscopy in neuroscience. J. Microsc. 259(2), 137-142 (2015). https://doi.org/10.1111/jmi.12244
  17. S.A. Wilke, J.K. Antonios, E.A. Bushong, A. Badkoobehi, E. Malek, M. Hwang, M. Terada, M.H. Ellisman, A. Ghosh, Deconstructing complexity: Serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse. J. Neurosci. 33(2), 507-522 (2013). https://doi.org/10.1523/JNEUROSCI.1600-12.2013