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Cited by 191Advanced Electron Microscopy Techniques and ApplicationsAdvanced Materials Characterization TechniquesNeuroscience and Neuropharmacology Research

Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction

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Merchan-Pérez, Angel·2009·Frontiers in Neuroanatomy
DOI 10.3389/neuro.05.018.2009PMC PMC2784681
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Abstract

The advent of transmission electron microscopy (TEM) in the 1950s represented a fundamental step in the study of neuronal circuits. The application of this technique soon led to the realization that the number of synapses changes during the course of normal life, as well as under certain pathological or experimental circumstances. Since then, one of the main goals in neurosciences has been to define simple and accurate methods to estimate the magnitude of these changes. Contrary to analysing single sections, TEM reconstructions are extremely time-consuming and difficult. Therefore, most quantitative studies use stereological methods to define the three-dimensional characteristics of synaptic junctions that are studied in two dimensions. Here, to count the exact number of synapses per unit of volume we have applied a new three-dimensional reconstruction method that involves the combination of focused ion beam milling and scanning electron microscopy (FIB/SEM). We show that the images obtained with FIB/SEM are similar to those obtained with TEM, but with the advantage that FIB/SEM permits serial reconstructions of large volumes of tissue to be generated rapidly and automatically. Furthermore, we compared the estimates of the number of synapses obtained with stereological methods with the values obtained by FIB/SEM reconstructions. We concluded that FIB/SEM not only provides the actual number of synapses per volume but it is also much easier and faster to use than other currently available TEM methods. More importantly, it also avoids most of the errors introduced by stereological methods and overcomes the difficulties associated with these techniques.

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  • Automated volume electron microscopy methods are a valuable and efficient approach for identifying synapses in three dimensions and have become the gold standard technique for this purpose.

    “Fortunately, the recent introduction of automated volume electron microscopy methods has proven to be a valuable and efficient approach for identifying synapses in three dimensions, becoming the gold standard technique for this purpose for this task (Merchán-Pérez et al., 2009).”

    From Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy (2024) · cites this paper as Merchán-Pérez et al., 2009

    The Introduction and Results (pages not available in our copy) describe automated FIB/SEM imaging that enables efficient three-dimensional identification and counting of synapses. The paper does not call it the gold standard, and its findings about FIB/SEM do not establish that status for automated volume electron microscopy methods generally. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) enables automated serial sectioning of large volumes of tissue, without any mechanical interaction with the sample.

    “This technology was chosen because it enables automated serial sectioning of large volumes of tissue, without any mechanical interaction with the sample (e.g., see Knott et al., 2008; Merchán-Pérez et al., 2009; Titze and Genoud, 2016; Kubota et al., 2018; Rollenhagen et al., 2020).”

    From Unambiguous identification of asymmetric and symmetric synapses using volume electron microscopy (2024) · cites this paper as Merchán-Pérez et al., 2009

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Research works printed but not linked: 2

  1. Colonnier M. (1981). The electron-microscopic analysis of the neuronal organization of the cerebral cortex. In The Organization of the Cerebral Cortex, Schmitt F. O. Worden F. G. Adelman G. Dennis S. G., eds (Cambridge, MA, MIT Press), pp. 125– 152
  2. Peters A. (1987). Synaptic specificity in the cerebral cortex. In Synaptic Function, Edelman G. M. Gall W. E. Cowan W. M., eds (New York, John Wiley), pp. 373– 397

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Authors
Merchan-Pérez, A.
Venue
Frontiers in Neuroanatomy
Publisher
Frontiers Media SA
Published
2009
Volume
3
Pages
18-18
ISSN
1662-5129 (ISSN-L)
DOI
10.3389/neuro.05.018.2009
PMC
PMC2784681
OpenAlex
W2063752629
Cited by
191
References
60
Citations by year
2026: 5 · 2025: 7 · 2024: 10 · 2023: 4 · 2022: 10 · 2021: 13 · 2020: 15 · 2019: 9 · 2018: 17 · 2017: 12 · 2016: 12 · 2015: 19 · 2014: 12 · 2013: 17 · 2012: 10
FWCI
5.56
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Neuroanatomy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • École Polytechnique Fédérale de Lausanne
  • Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Physical Sciences › Engineering › Biomedical Engineering › Advanced Materials Characterization Techniques
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
FIB-SEM, 3D reconstruction, stereology, synapses, synaptic density, serial sectioning, neural circuits, transmission electron microscopy

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