Sectioning
A rotary microtome advances a paraffin block a set distance past a fixed disposable blade on each turn, producing a ribbon of 3–5 µm sections that is floated flat on a warm water bath and picked up on slides.
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Microtome
A rotary microtome advances a paraffin block a set distance past a fixed disposable blade on each turn, producing a ribbon of 3–5 µm sections that is floated flat on a warm water bath and picked up on slides.
How it differs from the standard method
- Chill the block face between sections.
- Trim to a full face at 10–20 µm, then cut at the setting.
- Dry sections on before staining.
1Slices thin enough to see through
Light microscopy needs tissue thin enough for light to pass and for cells not to pile on top of each other. A nucleus is 5–10 µm across, so a 4 µm paraffin section holds roughly one layer of cells.
2What the knife does to the section
Every fault in a section was put there by the cut. If the wax is warm and soft, or the edge is dull, the knife pushes the tissue together before it slices it: the section comes out compressed along the cutting direction, with round nuclei squashed into ellipses.
Rotary microtome
Ribbons of 3–5 µm sections from a chilled wax block, flattened on a 40–45 °C water bath and dried onto charged slides.
Cryostat
A microtome inside a freezer. 5–10 µm sections from OCT blocks, at a temperature that suits the tissue.
Vibratome
A blade vibrating sideways slices soft tissue held in agarose; 20–400 µm sections for free-floating staining or live slices.
3Temperature is the hidden variable
Paraffin cuts best cold, so blocks are chilled face down on ice between sections. Frozen tissue has the opposite problem: each tissue has a narrow window where it is firm but not brittle.
4Vibrating blades and thick sections
A vibratome cuts by sideways vibration rather than pressure, so the blade must advance slowly compared with how far it swings. Fast advance and a small stroke push and tear the tissue, and the thickness wanders along the cut.
Key terms
Practise these as flashcards →The small angle (about 3–8°) between the back of the knife bevel and the block face; too small rubs, too large scrapes and chatters.
Shortening of the section along the cutting direction as the knife pushes the tissue; partly relaxed on a warm water bath.
Regular bands of thick and thin section parallel to the knife edge, caused by vibration of the knife or the block.
Tears running the length of the section in the cutting direction, from a nick or debris on the edge.
A glass plate set just above a cryostat knife edge so the section slides out flat instead of curling.
Touching a room-temperature slide to a frozen section so that it melts onto and sticks to the glass.
A thick section stained loose in wells of solution before mounting, common for brain.
When to use it
- Rotary microtome: routine paraffin sections for H&E, special stains and immunohistochemistry.
- Cryostat: frozen sections for lipids, enzymes, fragile epitopes or same-day results.
- Vibratome: thick sections of fixed brain for free-floating staining, or live acute slices for electrophysiology.
Compared with alternatives
A poorly processed block cannot be rescued at the microtome: soft centres crumble and over-hardened blocks chatter.
The usual way section quality is judged: compression, folds and chatter all show on H&E.
Electron microscopy needs ultrathin sections (50–90 nm) cut from resin blocks with glass or diamond knives.
Uses live vibratome slices, cut in ice-cold, oxygenated solution.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A rotary microtome advances a paraffin block a set distance past a fixed disposable blade on each turn, producing a ribbon of 3–5 µm sections that is floated flat on a warm water bath and picked up on slides.
- Chill the block face between sections.
- Trim to a full face at 10–20 µm, then cut at the setting.
- Dry sections on before staining.