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Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation.

Korogod N, Petersen CC, Knott GW - Elife (2015)

Bottom Line: However, this is known to cause significant tissue distortion including a reduction in the extracellular space.As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation.The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.

View Article: PubMed Central - PubMed

Affiliation: BioEM Facility, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT
Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.

No MeSH data available.


Related in: MedlinePlus

Comparison between chemical fixation (left hand images; A and C) and cryo fixation (right hand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amounts of extracellular space.However, this can only be clearly seen within 10 microns of the slice surface (B). Deeper into the cryo-fixed slice, the cellular elements appear disrupted with a fine latticed patterning indicating damage caused by ice crystal formation (D). This is not apparent at the same depth in the chemical fixed slice (C). Scale bar is 1 micron.DOI:http://dx.doi.org/10.7554/eLife.05793.006
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fig1s2: Comparison between chemical fixation (left hand images; A and C) and cryo fixation (right hand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amounts of extracellular space.However, this can only be clearly seen within 10 microns of the slice surface (B). Deeper into the cryo-fixed slice, the cellular elements appear disrupted with a fine latticed patterning indicating damage caused by ice crystal formation (D). This is not apparent at the same depth in the chemical fixed slice (C). Scale bar is 1 micron.DOI:http://dx.doi.org/10.7554/eLife.05793.006

Mentions: To check that the high-pressure freezing itself does not cause a significant alteration of the tissue morphology, we fixed acute slices, prepared in the same manner as for electrophysiological recording, with the cryo fixation or by immersion in the chemical fixative. Both groups showed similar tissue quality and levels of extracellular space, at least where ice crystal formation had not disrupted the ultrastructure (Figure 1—figure supplement 2).


Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation.

Korogod N, Petersen CC, Knott GW - Elife (2015)

Comparison between chemical fixation (left hand images; A and C) and cryo fixation (right hand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amounts of extracellular space.However, this can only be clearly seen within 10 microns of the slice surface (B). Deeper into the cryo-fixed slice, the cellular elements appear disrupted with a fine latticed patterning indicating damage caused by ice crystal formation (D). This is not apparent at the same depth in the chemical fixed slice (C). Scale bar is 1 micron.DOI:http://dx.doi.org/10.7554/eLife.05793.006
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4530226&req=5

fig1s2: Comparison between chemical fixation (left hand images; A and C) and cryo fixation (right hand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amounts of extracellular space.However, this can only be clearly seen within 10 microns of the slice surface (B). Deeper into the cryo-fixed slice, the cellular elements appear disrupted with a fine latticed patterning indicating damage caused by ice crystal formation (D). This is not apparent at the same depth in the chemical fixed slice (C). Scale bar is 1 micron.DOI:http://dx.doi.org/10.7554/eLife.05793.006
Mentions: To check that the high-pressure freezing itself does not cause a significant alteration of the tissue morphology, we fixed acute slices, prepared in the same manner as for electrophysiological recording, with the cryo fixation or by immersion in the chemical fixative. Both groups showed similar tissue quality and levels of extracellular space, at least where ice crystal formation had not disrupted the ultrastructure (Figure 1—figure supplement 2).

Bottom Line: However, this is known to cause significant tissue distortion including a reduction in the extracellular space.As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation.The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.

View Article: PubMed Central - PubMed

Affiliation: BioEM Facility, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT
Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.

No MeSH data available.


Related in: MedlinePlus