Specimen Transfer and Cryosectioning
Specimen Transfer to Pre-cooled Cryochamber.
To cut thin sections for electron microscopy, temperatures between -90°C and -140°C are recommended. Semi-thin sections (0.2-1.0 mm) can be cut at around -90°C. Remember, the colder the temperature, the harder the block, and vice versa.
Transfer the specimen, on the specimen stub, from the liquid nitrogen using pre-cooled forceps and quickly place it into the pre-cooled cryochamber. Place the specimen stub into the specimen holder and lock it in place. Any tools inserted into the cryochamber, and especially those which are used to manipulate the specimen, must be pre-cooled in liquid nitrogen.
All the commercially available cryo ultramicrotomes can be easily and routinely used for cryosectioning.
The Knife.
The most important factor to obtaining good cryosections is the quality of the knife. The knives used on this course have been made by carefully breaking glass strips. Knife-making machines are available and the theory behind glass knife making has been covered in great detail before (Griffiths et al., 1983). Until recently glass knives were the preferred tool for production of cryosections. Recent developments in diamond knife production, however, has meant that these can be used routinely for the production of good cryosections.
Preparing the Block for Sectioning.
It is possible to cut sections from a block of any shape but it is easier to obtain sections from blocks that have been trimmed prior to sectioning. This can be easily done with a trimming tool, a scalpel or with a glass knife.
If using a trimming tool or glass knife, carefully advance the cutting edge to the block face and commence facing the block. This is done by taking off sections from the block surface until it is flat. If the sections are too thick the block may fracture resulting in an unpolished surface or even loss of the block.
Once the surface is flat, the sides can be trimmed so as to give the block a square or oblong shape. It is important to make the bottom edge parallel with the knife edge and the vertical sides perpendicular to the knife edge.
Sectioning
Once the block has been trimmed to shape, it can be carefully advanced to an unused part of the knife and sectioned. Care should be taken when bringing the knife and the block together to avoid cutting a thick first section. Again this may ruin, or loosen, the block.
Use the rough advance to bring the knife close to the block (about 1-2 mm away) then use the fine advance to either move the knife or the block. When it seems as if the knife and the block are almost touching, start sectioning by hand, using the specimen advance, to bring the knife and the block together. Switch over to automatic sectioning when sections appear on the knife.
It is recommended to use a slow cutting speed for although melting does not appear to occur, various other cutting artifacts may appear (Chang et al, 1983). The slow cutting speed may be combined with a fast return cycle in order to reduce the possibility of thermal changes occurring in the specimen.
Manipulating the sections.
Once sections have been obtained, unless using an anti-static device, the sections will lie on the knife surface close to the edge. They can be carefully moved away using an eyelash mounted on a stick. It is important to separate sections that are lying on top of each other and to arrange them into small separate groups ready for picking up. When using the eyelash it may be best to hold the sections down rather than trying to move them with the eyelash positioned under them.
The cryochamber is cooled to -120°C and left to stabilize at this temperature. The specimen block is then carefully brought up to the knife (in this example it is a diamond knife) and sections obtained. Although difficult to see, they have interference colors, the frozen sections gather on the surface of the knife. The trim tool, used to shape the block before sectioning, is on the right of the picture |
