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


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 ( 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.

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Figure 1: Two-photon in vivo imaging and fiducial laser-branding marks.
Figure 2: Block preparation for the FIBSEM.
Figure 3: Imaging the region of interest with FIBSEM.
Figure 4: Segmentation and 3D reconstruction.


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This work was supported by the Swiss National Foundation Synergia project grant CRF II313470/1 (G.W.K.) and project grant 31003A_135631 (A.H.).

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G.W.K. and A.H. conceived and designed experiments. A.H., G.W.K. and B.M. performed in vivo imaging, vibratome sectioning and laser mark branding. G.W.K., M.C. and B.M. performed electron microscopy and FIBSEM imaging. A.H., G.W.K., B.M., A.K. and F.A.H. analyzed the data and materials, and contributed reagents and analysis tools. G.W.K., B.M., A.H., M.C. and F.A.H. wrote the manuscript.

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Correspondence to Graham W Knott.

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The authors declare no competing financial interests.

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Maco, B., Cantoni, M., Holtmaat, A. et al. Semiautomated correlative 3D electron microscopy of in vivo–imaged axons and dendrites. Nat Protoc 9, 1354–1366 (2014).

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