Two- and three-dimensional electron microscopy techniques: powerful tools for studying the brain under physiological and pathological conditions

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Abstract
We are in the midst of a revolution in the fields of neuroanatomy and electron microscopy. The monumental advancements in the neuroscience field during the last decade have led to unprecedented scientific discoveries about our brain and to the development of new technologies and applications that have significantly contributed to such advances. Conventional applications of transmission electron microscopy have revolutionized neurosciences and are critical for determining the fine morpho-functional characterization of brain cells and their connections. Electron microscopy has progressively evolved toward the development of both more sensitive approaches to unravel the bidimensional subcellular localization of proteins and tools that allow for the three-dimensional characterization of different nerve cells and their connections. The development of new technological advances in two- and three-dimensional electron microscopy to study and map the brain has led to the development of essential tools to decipher the complexity of the brain. For two-dimensional, the sodium dodecyl sulfate-digested freeze-fracture replica labeling technique is a technique with the main goal of chemically identifying the structural components viewed in freeze-fracture replicas and has significant advantages over conventional immunoelectron microscopic techniques for revealing the subcellular organization of proteins along the neuronal surface in the brain. For three-dimensional, volume electron microscopy methods can be applied to structural studies of cell components and organelles, just as conventional transmission electron microscopy has been traditionally applied, but with advantages derived from the possibility of three-dimensional visualization and analysis. The development of volume electron microscopy has greatly facilitated the study of brain structure and connectivity at the synaptic level. Dedicated software tools for the analysis of highly complex connectivity patterns in three dimension are evolving in parallel, allowing the extraction of relevant information from large datasets. Moreover, by applying these new methodologies, the field of pathology is expected to advance, potentially with the identification of the pathogenesis generating these diseases. This review aims to present the possibilities and fundamentals of two- and three-dimensional electron microscopy for high-resolution ultrastructural analyses of neurons and their connections. These technological tools have improved the ability to study the brain, thus providing new insights into brain structure and function.

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