Scanned Electron Microscopy Sample Preparation workshop

Instruments for surface preparation and fine sectioning of biological samples
 

• Vibratome

Used for fine sectioning or surface trimming of “soft” samples (e.g., leaves, roots). Samples are embedded in a medium (agar-agar) that holds them in position and presents the surface for cutting. A razor blade, positioned at an adjustable angle and in contact with the surface, moves with a calibrated vibration frequency to slice the sample into thin sections. After each cut, the sample stage moves vertically (adjustable) by the desired section thickness (a few micrometres) to re-engage with the blade. Wood sample sectioning is limited to small twigs due to the material’s rigidity.

• Microtomes

For more information, see the “Histological Microscopy Preparation Unit” page.

• Sliding microtome for wood

Equipped with interchangeable clamps to hold tree core segments, this instrument uses a disposable blade with adjustable inclination and cutting angles to produce micrometric sections or surface trimming. The section thickness is adjusted between passes via a micrometric vertical control wheel that moves the specimen stage towards the blade.

• Core microtome for wood

Derived from the sliding microtome, this version allows surface trimming (for dendrometry, dendrochemistry, etc.) or sectioning of a full-length core (several tens of cm) using the same operating principles.

These instruments were developed by WSL in Switzerland: Watch the video

• Cryo-microtome / Cryostat (Leica CM3050S)

The cutting principle is similar to that of the microtomes described above: a disposable blade, set at specific attack and inclination angles, slices the specimen. Here, the specimen is frozen and embedded in a medium that polymerises at low temperature (e.g., Tissue-Tek® at –10 °C). Both the specimen holder and blade holder can be cooled to –30 °C. Sectioning can be performed manually (wheel or hand crank) or motorised (series of programmed cuts).

Freeze Dryer with Cooled Shelves and Controlled Pressure
 

• Labconco FreeZone 6 L Benchtop, Stoppering Tray Dryer, 3 shelves

Before SEM observations or analyses under high vacuum (~10⁻⁴ Pa), hydrated samples must either be frozen and introduced into the SEM chamber via a cryogenic transfer system with a cooled stage (–140 °C) or dehydrated to allow high-magnification, high-resolution surface imaging. Air-drying often causes excessive artefacts or deformations, making proper observation impossible.

Freeze-drying techniques have long been developed on the facility to: reduce deformation from drying of raw samples (e.g., wood cores, discs) and preserve mineral elements of interest for microanalysis within the tissues.

This system removes water (ice) slowly and in a controlled manner by adjusting chamber pressure (down to a few hundred Pa) while samples remain frozen on cooled shelves (temperature adjustable and programmable).

Freeze-drying phases:

• Primary drying: Rapid sublimation of surface ice crystals and free water within the sample, occurring mainly during the first hours under vacuum while shelves are kept cold (organic compounds remain unaltered).
• Secondary drying: Much slower and dependent on sample type and bound water content within tissues. Controlled warming ramps with temperature plateaus remove this bound water through slow sublimation.
• Final phase: Shelf temperature is raised to ambient or slightly above before re-pressurisation to atmospheric conditions, preventing rehydration from ambient humidity upon opening.

Contact:

• Equipments coordinator: Maryline Harroué maryline.harroue@inra.fr 
• Unit coordinator: Julien Ruelle julien.ruelle@inrae.fr