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Specimen Preparation Techniques for EM

Visualizing a biological specimen with an electron microscope is not a trivial task, mostly because of the intrinsic nature of the electron and matter interactions that are responsible for the image formation. In order to be imaged using TEM the specimen has to be very thin and for using SEM the specimen needs a conductive surface. In both cases the specimen has to be placed inside high vacuum. Thus, biological specimens cannot be imaged in their native state and need to be heavily processed. Accordingly, sample preparation is a crucial step, which represents an essential activity and service in our facility.

For ultrastructure

The focus of these preparations is to provide best possible preservation of the specimen (fixation, dehydration and embedding) and to facilitate access to the intracellular structures (sectioning).

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Negative staining

Negative staining is a rapid method for screening the morphology, size and arrangement of particulate samples such as…

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High pressure freezing and freeze substitution

To avoid the occurrence of artifacts, which can be introduced by chemical fixation, a sample can instead be vitrified under…

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Chemical fixation and epoxy embedding

The chemical fixation and epoxy embedding techniques are used for conventional TEM imaging, 3D electron tomography and…

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High throughput serial sectioning

In addition to conventional resin block sectioning and cryosectioning our facility is equipped with tools for high…

For immunodetection

Immuno-preparations aim to preserve, expose and detect epitopes, in some cases at a cost to ultrastructure. The samples are crosslinked weekly or cryo-immobilised and processed more gently; solvent dehydration is omitted or performed at sub-zero temperatures and resin is cured with UV light instead of heat. The epitopes are detected using 1° antibodies and 2° probes tagged with gold particles.

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Tokuyasu technique

The Tokuyasu technique involves neither sample dehydration with solvents nor resin-embedding. This means that antigens…

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Chemical fixation and acrylic resin embedding

This technique combines gentle chemical fixation with embedding in a hydrophilic acrylic resin such as LR Gold.

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High pressure freezing and freeze substitution

To avoid the occurrence of artifacts, which can be introduced by chemical fixation, a sample can instead be vitrified under…

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Pre-embedding labelling

This technique is based on introducing the primary and secondary antibodies with gold tags into the specimen while it is…

For SEM topography

Similarly to the ultrastructure preparations described above this technique ensures the preservation of fine ultrastructural details and stabilisation of the specimen. However, the specimen is not in any way embedded. Since charging is much more of an issue than in TEM it is crucial to render the specimen conductive to avoid image distortions.

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SEM topography preparation

Plunge-freezing

At our facility the plunge-freezing technique is used primarily for cryo-screening of particulate samples.

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Plunge-freezing