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Semiautomated correlative 3D electron microscopy of in vivo–imaged axons and dendrites

Kreshuk, Anna
Hamprecht, Fred A
Knott, Graham W
Published in Nature Protocols. 2014, vol. 9, no. 6, p. 1354-1366
Abstract This protocol describes how in vivo-imaged dendrites and axons in adult mouse brains can subsequently be prepared and imaged with focused ion beam scanning electron microscopy (FIBSEM). The procedure starts after in vivo imaging with chemical fixation, followed by the identification of the fluorescent structures of interest. Their position is then highlighted in the fixed tissue by burning fiducial marks with the two-photon laser. Once the section has been stained and resin-embedded, a small block is trimmed close to these marks. Serially aligned EM images are acquired through this region, using FIBSEM, and the neurites of interest are then reconstructed semiautomatically by using the ilastik software (http://ilastik.org/). This reliable imaging and reconstruction technique avoids the use of specific labels to identify the structures of interest in the electron microscope, enabling optimal chemical fixation techniques to be applied and providing the best possible structural preservation for 3D analysis. The entire protocol takes ∼4 d.
PMID: 24833174
Full text
Research group Les circuits neuronaux dans le néocortex des mammifères (855)
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MACO, Bohumil et al. Semiautomated correlative 3D electron microscopy of in vivo–imaged axons and dendrites. In: Nature Protocols, 2014, vol. 9, n° 6, p. 1354-1366. https://archive-ouverte.unige.ch/unige:40893

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Deposited on : 2014-10-15

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