Abstract
FIB-SEM is an electron microscopy technique that allows the acquisition of serial sections in an automated manner. A Focused Ion Beam (FIB) is directed toward the specimen, removing material from its surface. Since the FIB can be positioned and controlled on a nanometer scale, the specimen surface can be milled so that a thin layer of a specified thickness is removed. The Scanning Electron Microscope (SEM) column is then used to acquire an image from the freshly milled surface. To obtain a series of images, the milling/imaging cycle is repeated automatically. This technique can be used with any pre-embedding method that yields an electron-dense end product. We describe here an immunocytochemical protocol to be used on vibratome brain sections. After the immunocytochemical procedure, the sections are flat embedded in epoxy resin and prepared for FIB-SEM imaging. Serial image acquisition, visualization, and analysis in 3D are also briefly described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stevens JK, Davis TL, Friedman N, Sterling P (1980) A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections. Brain Res 2:265–293. https://doi.org/10.1016/0165-0173(80)90010-7
Harris KM, Perry E, Bourne J et al (2006) Uniform serial sectioning for transmission electron microscopy. J Neurosci 26:12101–12103. https://doi.org/10.1523/JNEUROSCI.3994-06.2006
Hoffpauir BK, Pope BA, Spirou GA (2007) Serial sectioning and electron microscopy of large tissue volumes for 3D analysis and reconstruction: a case study of the calyx of held. Nat Protoc 2:9–22. https://doi.org/10.1038/nprot.2007.9
Bock DD, Lee W-CA, Kerlin AM et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182. https://doi.org/10.1038/nature09802
Lee TJ, Kumar A, Balwani AH et al (2018) Large-scale neuroanatomy using LASSO: loop-based automated serial sectioning operation. PLoS One 13:e0206172. https://doi.org/10.1371/journal.pone.0206172
Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329. https://doi.org/10.1371/journal.pbio.0020329
Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28:2959–2964. https://doi.org/10.1523/JNEUROSCI.3189-07.2008
Merchán-Pérez A, Rodriguez J-R, Alonso-Nanclares L et al (2009) Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction. Front Neuroanat 3:18. https://doi.org/10.3389/neuro.05.018.2009
Kasthuri N, Hayworth KJ, Berger DR et al (2015) Saturated reconstruction of a volume of neocortex. Cell 162:648–661. https://doi.org/10.1016/j.cell.2015.06.054
Kubota Y, Sohn J, Kawaguchi Y (2018) Large volume electron microscopy and neural microcircuit analysis. Front Neural Circuits 12:98. https://doi.org/10.3389/fncir.2018.00098
Helmstaedter M (2013) Cellular-resolution connectomics: challenges of dense neural circuit reconstruction. Nat Methods 10:501–507. https://doi.org/10.1038/nmeth.2476
Langford RM (2006) Focused ion beams techniques for nanomaterials characterization. Microsc Res Tech 69:538–549. https://doi.org/10.1002/jemt.20324
Anton-Sanchez L, Bielza C, Merchan-Perez A et al (2014) Three-dimensional distribution of cortical synapses: a replicated point pattern-based analysis. Front Neuroanat 8:85. https://doi.org/10.3389/fnana.2014.00085
Merchán-Pérez A, Rodríguez J-R, González S et al (2014) Three-dimensional spatial distribution of synapses in the neocortex: a dual-beam electron microscopy study. Cereb Cortex 24:1579–1588. https://doi.org/10.1093/cercor/bht018
Santuy A, Rodriguez J-R, DeFelipe J, Merchan-Perez A (2018) Volume electron microscopy of the distribution of synapses in the neuropil of the juvenile rat somatosensory cortex. Brain Struct Funct 223:77–90. https://doi.org/10.1007/s00429-017-1470-7
Santuy A, Rodriguez J-R, DeFelipe J, Merchan-Perez A (2018) Study of the size and shape of synapses in the juvenile rat somatosensory cortex with 3D electron microscopy. eNeuro 5:e0377–17.2017. https://doi.org/10.1523/ENEURO.0377-17.2017
Santuy A, Turégano-López M, Rodríguez JR et al (2018) A quantitative study on the distribution of mitochondria in the neuropil of the juvenile rat somatosensory cortex. Cereb Cortex 28:3673–3684. https://doi.org/10.1093/cercor/bhy159
Turegano-Lopez M, Santuy A, DeFelipe J, Merchan-Perez A (2020) Size, shape, and distribution of multivesicular bodies in the juvenile rat somatosensory cortex: a 3D electron microscopy study. Cereb Cortex 30:1887–1901. https://doi.org/10.1093/cercor/bhz211
Domínguez-Álvaro M, Montero-Crespo M, Blazquez-Llorca L et al (2018) Three-dimensional analysis of synapses in the transentorhinal cortex of Alzheimer’s disease patients. Acta Neuropathol Commun 6:20. https://doi.org/10.1186/s40478-018-0520-6
Domínguez-Álvaro M, Montero-Crespo M, Blazquez-Llorca L et al (2019) 3D electron microscopy study of synaptic Organization of the Normal Human Transentorhinal Cortex and its Possible Alterations in Alzheimer’s disease. eNeuro 6. https://doi.org/10.1523/ENEURO.0140-19.2019
Montero-Crespo M, Domínguez-Álvaro M, Rondón-Carrillo P, et al (2020) Three-dimensional synaptic organization of the human hippocampal CA1 field. bioRxiv 2020.02.25.964080. https://doi.org/10.1101/2020.02.25.964080
Bosch C, Martínez A, Masachs N et al (2015) FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons. Front Neuroanat 9:60. https://doi.org/10.3389/fnana.2015.00060
Bosch C, Masachs N, Exposito-Alonso D et al (2016) Reelin regulates the maturation of dendritic spines, synaptogenesis and glial Ensheathment of newborn granule cells. Cereb Cortex 26:4282–4298. https://doi.org/10.1093/cercor/bhw216
Rodriguez-Moreno J, Rollenhagen A, Arlandis J et al (2018) Quantitative 3D ultrastructure of Thalamocortical synapses from the “Lemniscal” ventral posteromedial nucleus in mouse barrel cortex. Cereb Cortex 28:3159–3175. https://doi.org/10.1093/cercor/bhx187
Rodriguez-Moreno J, Porrero C, Rollenhagen A et al (2020) Area-specific synapse structure in branched posterior nucleus axons reveals a new level of complexity in Thalamocortical networks. J Neurosci 40:2663–2679. https://doi.org/10.1523/JNEUROSCI.2886-19.2020
Kikuchi T, Gonzalez-Soriano J, Kastanauskaite A et al (2020) Volume electron microscopy study of the relationship between synapses and astrocytes in the developing rat somatosensory cortex. Cereb Cortex 30:3800–3819. https://doi.org/10.1093/cercor/bhz343
Morales J, Alonso-Nanclares L, Rodríguez J-R et al (2011) Espina: a tool for the automated segmentation and counting of synapses in large stacks of electron microscopy images. Front Neuroanat 5:18. https://doi.org/10.3389/fnana.2011.00018
Gray EG (1959) Axo-somatic and axo-dendritic synapses of the cerebral cortex: an electron microscope study. J Anat 93:420–433
Colonnier M (1968) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res 9:268–287
Howard CV, Reed MG (2005) Unbiased stereology : three-dimensional measurement in microscopy, 2nd edn. Garland Science/BIOS Scientific Publishers, Oxon, UK
Morales J, Rodríguez A, Rodríguez J-R et al (2013) Characterization and extraction of the synaptic apposition surface for synaptic geometry analysis. Front Neuroanat 7:20. https://doi.org/10.3389/fnana.2013.00020
Rodríguez J-R, Turégano-López M, DeFelipe J, Merchán-Pérez A (2018) Neuroanatomy from mesoscopic to nanoscopic scales: an improved method for the observation of semithin sections by high-resolution scanning electron microscopy. Front Neuroanat 12:14. https://doi.org/10.3389/fnana.2018.00014
Acknowledgments
The authors would like to thank L. Valdés and M.C. Álvarez for their technical assistance and Nick Guthrie for his excellent text editing. This work was partially supported by grants from the following entities: Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0066, Spain); and the Spanish Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-094307-B-I00 and the Cajal Blue Brain Project [the Spanish partner of the Blue Brain Project initiative from EPFL, Switzerland]).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Turégano-López, M., Rodríguez, JR., Alonso-Nanclares, L., González-Soriano, J., DeFelipe, J., Merchán-Pérez, A. (2021). Pre-Embedding Immunostaining of Brain Tissue and Three-Dimensional Imaging with FIB-SEM. In: Lujan, R., Ciruela, F. (eds) Receptor and Ion Channel Detection in the Brain. Neuromethods, vol 169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1522-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1522-5_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1521-8
Online ISBN: 978-1-0716-1522-5
eBook Packages: Springer Protocols