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Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons.

Maco B, Holtmaat A, Cantoni M, Kreshuk A, Straehle CN, Hamprecht FA, Knott GW - PLoS ONE (2013)

Bottom Line: Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information.These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms.The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

View Article: PubMed Central - PubMed

Affiliation: BioEM Facility, Centre of Electron Microscopy, EPFL, Lausanne, Switzerland.

ABSTRACT
Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

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In vivo imaging, laser branding and tissue preparation.A, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (B), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (C) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (D) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (E). This indicates the region to be imaged (F) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in A and B is 100 µm, and 10 µm in (C–F).
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pone-0057405-g001: In vivo imaging, laser branding and tissue preparation.A, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (B), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (C) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (D) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (E). This indicates the region to be imaged (F) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in A and B is 100 µm, and 10 µm in (C–F).

Mentions: After perfusion fixation, the imaged brain region was then sliced tangentially to the cortical surface, parallel to the imaging window and focal plane of the 2-photon laser-scanning microscopy (2PLSM). This ensured that the first few sections contained the region of interest, as well as the brain surface vasculature, as it was seen through the cranial window (Fig. 1A) in the live animal. This vasculature provided essential landmarks with which the fluorescent neurites of interest could be located in the fixed sections (Fig. 1A, B). Capturing the vascular pattern is critically important to pinpoint imaged axons and dendrites, particularly in neural tissue that shows high densities of fluorescent neurons.


Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons.

Maco B, Holtmaat A, Cantoni M, Kreshuk A, Straehle CN, Hamprecht FA, Knott GW - PLoS ONE (2013)

In vivo imaging, laser branding and tissue preparation.A, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (B), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (C) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (D) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (E). This indicates the region to be imaged (F) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in A and B is 100 µm, and 10 µm in (C–F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585404&req=5

pone-0057405-g001: In vivo imaging, laser branding and tissue preparation.A, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (B), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (C) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (D) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (E). This indicates the region to be imaged (F) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in A and B is 100 µm, and 10 µm in (C–F).
Mentions: After perfusion fixation, the imaged brain region was then sliced tangentially to the cortical surface, parallel to the imaging window and focal plane of the 2-photon laser-scanning microscopy (2PLSM). This ensured that the first few sections contained the region of interest, as well as the brain surface vasculature, as it was seen through the cranial window (Fig. 1A) in the live animal. This vasculature provided essential landmarks with which the fluorescent neurites of interest could be located in the fixed sections (Fig. 1A, B). Capturing the vascular pattern is critically important to pinpoint imaged axons and dendrites, particularly in neural tissue that shows high densities of fluorescent neurons.

Bottom Line: Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information.These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms.The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

View Article: PubMed Central - PubMed

Affiliation: BioEM Facility, Centre of Electron Microscopy, EPFL, Lausanne, Switzerland.

ABSTRACT
Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

Show MeSH