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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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Related in: MedlinePlus

Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.A, B, The FIBSEM image series can be used to segment in vivo imaged structures (A, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (B, inset left) that also includes the synaptically coupled dendritic spine (B, inset right). Scale bar in A is 1 µm, and in A (inset) is 5 µm.
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pone-0057405-g003: Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.A, B, The FIBSEM image series can be used to segment in vivo imaged structures (A, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (B, inset left) that also includes the synaptically coupled dendritic spine (B, inset right). Scale bar in A is 1 µm, and in A (inset) is 5 µm.

Mentions: A detailed manual segmentation was carried out on a single glutamatergic (presumed) bouton seen in the live brain (Fig. 3). Using immunocytochemistry methods to identify such a structure would leave its morphology compromised, with poorly preserved membranes and features concealed by the labeling. Here, with no specific labeling, the quality of the near isotropic imaging allows all the membranes and organelles to be seen. The size of the total imaged volume at this resolution also contains the synaptically connected dendrite that has been included in the model (Fig. 3). The entire reconstruction was made from a sub-volume of the original image stack with dimensions of 6.40 µm×8.0 µm ×5.0 µm. A single section of this thickness would be impossible to image with any transmission electron tomography (ET) method. Targeting a specific site like this for ET would also be hindered by the use of thin sections manually cut and placed onto supporting grids where the metal bars may obscure the required regions. Achieving such a 3D resolution would also not be possible with serial section TEM in which section thicknesses are usually around 50 nm.


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)

Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.A, B, The FIBSEM image series can be used to segment in vivo imaged structures (A, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (B, inset left) that also includes the synaptically coupled dendritic spine (B, inset right). Scale bar in A is 1 µm, and in A (inset) is 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057405-g003: Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.A, B, The FIBSEM image series can be used to segment in vivo imaged structures (A, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (B, inset left) that also includes the synaptically coupled dendritic spine (B, inset right). Scale bar in A is 1 µm, and in A (inset) is 5 µm.
Mentions: A detailed manual segmentation was carried out on a single glutamatergic (presumed) bouton seen in the live brain (Fig. 3). Using immunocytochemistry methods to identify such a structure would leave its morphology compromised, with poorly preserved membranes and features concealed by the labeling. Here, with no specific labeling, the quality of the near isotropic imaging allows all the membranes and organelles to be seen. The size of the total imaged volume at this resolution also contains the synaptically connected dendrite that has been included in the model (Fig. 3). The entire reconstruction was made from a sub-volume of the original image stack with dimensions of 6.40 µm×8.0 µm ×5.0 µm. A single section of this thickness would be impossible to image with any transmission electron tomography (ET) method. Targeting a specific site like this for ET would also be hindered by the use of thin sections manually cut and placed onto supporting grids where the metal bars may obscure the required regions. Achieving such a 3D resolution would also not be possible with serial section TEM in which section thicknesses are usually around 50 nm.

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
Related in: MedlinePlus