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Advancing Array Tomography to Study the Fine Ultrastructure of Identified Neurons in Zebrafish (Danio rerio )

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Part of the Neuromethods book series (NM,volume 155)

Abstract

Array tomography (AT) provides a versatile workflow for correlated light and electron microscopy (CLEM). In short, biological tissues are embedded in EM-resins for immunolabeling, cut in ultrathin section arrays, which are mounted on glass slides, labeled and imaged for immunofluorescence at the light microscope and then prepared for scanning electron microscopy (SEM) imaging. Light- and electron micrographs obtained from the identical regions of interest of the same sections are then correlated to an aligned composite image series. We adapted this protocol to identify and image the Mauthner neuron of the developing zebrafish embryo. The Mauthner neuron is an identifiable neuron, which can be easily labeled by retrograde tracing with for example rhodamine dextran. We take advantage of the fact that the fluorescence of rhodamine is retained after embedding in the LR White resin. Furthermore, we expanded the workflow to reach a near-to-native ultrastructural preservation and good antigenicity of the nervous tissue, by applying high pressure freezing and freeze substitution. Moreover, we add structured illumination microcopy (SIM) as imaging modality to allow tracing of fine neuronal projections and increase correlation accuracy.

Key words

  • Array tomography
  • Zebrafish
  • Danio rerio
  • Mauthner neuron
  • Reticulospinal neuron
  • Identified neuron

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  • DOI: 10.1007/978-1-0716-0691-9_4
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Acknowledgments

We want to thank Manfred Schartl for generously providing us access to the fish facility. Furthermore, we thank Georg Krohne, Markus Engstler, Sebastian Markert, Markus Sauer, Marcus Behringer, Jean-Louis Bessereau, Camilla Luccardini, and Hong Zhan for many supportive discussions throughout the different stages of the project. We thank Claudia Gehrig, Daniela Bunsen, and Brigitte Trost for excellent technical support. We would like to thank Swarnima Joshi and Felix Erwin for their contribution during early stages of this project. This project was supported by the Universitätsbund Würzburg (AZ14-48). C.L. was funded by the Bayerische Gleichstellungsförderung.

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Correspondence to Christina Lillesaar or Christian Stigloher .

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Strobel, M., Helmprobst, F., Pauli, M., Heckmann, M., Lillesaar, C., Stigloher, C. (2020). Advancing Array Tomography to Study the Fine Ultrastructure of Identified Neurons in Zebrafish (Danio rerio ). In: Wacker, I., Hummel, E., Burgold, S., Schröder, R. (eds) Volume Microscopy . Neuromethods, vol 155. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0691-9_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0691-9_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0690-2

  • Online ISBN: 978-1-0716-0691-9

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