TISSUE SECTIONING

Trimming
The Microtome
• Sharp razor - earliest form of microtomy
• Modern microtomes are precision instruments
designed to cut uniformly thin sections of a variety
of materials for detailed microscopic examination.
– 1,800x, the thickness of a section (1 and 10
microns or thin sections
– For EM, several hundred thousands
magnification (10 nm or ultra-thin sections)
Parts of the Microtome
1. Base (microtome body)

2. Knife attachment and knife

3. Material or tissue holder

Types of Microtome
• ROTARY MICROTOME
– general purpose microtomes (also found in
cryostat)
Types of Microtome
• ROTARY MICROTOME
– for cutting semi-thin to thin sections for
light microscopy
– Rotary action of a hand wheel to activate
the advancement of a block towards a
rigidly held knife. The block moves up
and down in a vertical plane in relation to
the knife and therefore cuts flat sections.
– Lightweight: Suitable for cutting paraffin
-embedded mat’l in a continuous ribbon
Types of Microtome
• ROTARY MICROTOME
– Heavyweight: motor driven instruments used with
a slow, continuous speed and retracting advance
movement to section hard material embedded in
synthetic resin.
– Section thickness settings range from 0.5µm to
60µm on most machines.
– Sections of paraffin wax embedded tissues are
normally cut within the range 3 to 5µm whilst
resin sections are between 0.5 to 1µm.
– Usually used with disposable steel knives
Types of Microtome
• SLIDING MICROTOME
Types of Microtome
• SLIDING MICROTOME
– well suited to cutting a wide range of materials
including bone, plastics, resins and wood as well
as large area soft tissues
– Thick sections
Types of Microtome
• SLEDGE MICROTOME
Types of Microtome
• SLEDGE MICROTOME
– for cutting large blocks of paraffin and
resin embedded material including whole
organs, for light microscopy
– The knife holding clamps allow the knife
to be offset to the direction of cut, a
major advantage when sectioning large,
hard blocks.
Types of Microtome
• SLEDGE MICROTOME
– very heavy for stability and not usually
subject to vibration
– can also be used to cut materials from
various industrial applications (wood,
plastics, textile fibers)
– not suitable for cutting very hard resins
such as araldite because of the risk of
vibration
Types of Microtome
• FREEZING MICROTOME
Types of Microtome
• FREEZING MICROTOME
– for cutting thin to semi-thin sections of fresh,
frozen tissue and semi-thin sections from
industrial products such as some textiles, paper,
leather, soft plastics, rubber, powders, pastes
and food products
– equipped with a stage upon which tissue can be
quickly frozen using either liquid carbon
dioxide, from a cylinder, or a low temperature
recirculating coolant.
Types of Microtome
• FREEZING MICROTOME
– Some cooling systems also allow the knife to be
cooled at the same time.
– Note: the knife moves while the tissue block
remains static.
– The block moves by a pre-set amount, in
microns, at the end of each cut.
– Consistent, high quality, thin sections are very
difficult to obtain with this type of microtome.
Types of Microtome
• ULTRAMICROTOME
Types of Microtome
• ULTRAMICROTOME
Types of Microtome
• ULTRAMICROTOME
– used to prepare ultrathin sections for
light and electron microscopy.
– Very small samples of tissue or industrial
products are usually embedded in hard
resin before cutting.
– It has been reported that sections can be
cut as thin as 10 nanometres.
Types of Microtome
• ULTRAMICROTOME
– Two forms of advance mechanism
• thermal mechanism relies upon heat induced expansion
in a bifurcated metal strip
• mechanical form - a microprocessor coupled to a
precise stepping motor controls the advance
mechanism. The cutting stroke is motor driven to
provide a regular, smooth motion for sections of even
thickness and constant reproducibility. Knives are
usually made from glass, diamond or sapphire. The
block is brought to the knife edge under microscopical
control and as each section is cut it is floated on to a
water bath adjacent to the knife edge.
Types of Microtome
• CRYOSTAT
Types of Microtome
• CRYOSTAT
– primarily used for cutting sections of frozen
tissue as well as pastes, powders and some food
substances.
– commonly consists of a microtome contained
within a refrigerated chamber, the temperature
of which can be maintained at a preset level.
– A recent innovation has the body of the
microtome positioned outside the refrigerated
chamber.
Types of Microtome
• CRYOSTAT
– usually contains a rotary microtome although
some portable units utilise a rocking
microtome. With the object, object holder and
knife all at the same temperature and all other
conditions for cutting the material optimal,
sections as thin as 1 micron are possible.
Types of Microtome
• SAW MICROTOME
Types of Microtome
• SAW MICROTOME
– cut sections from very hard material such as
undecalcified bone, glass or ceramics.
– Samples (commonly embedded in resins) are
moved extremely slowly against a diamond
coated saw rotating at approximately 600 rpm.
– Sections of 20 µm or greater are possible
providing the saw blade is in perfect condition.
– Very thin sections are not possible.
Types of Microtome
• VIBRATING MICROTOME
Types of Microtome
• VIBRATING MICROTOME
– Originally conceived as a microtome which
could produce high quality sections of fresh,
unfixed material from animal or botanical
sources and to replace the hand microtome.
– The name of the instrument derives from the
high speed vibration produced in a safety razor
blade to provide the cutting power.
Types of Microtome
• VIBRATING MICROTOME
– The amplitude of vibration is adjusted by
altering electrical voltage applied to the 'knife'.
Different degrees of vibration are required to
produce sections from varying densities of
material.
– To prevent tearing, soft material is cut while
immersed in a fluid which also aids in
dissipating heat produced at the vibrating edge
of the razor as it cuts.
Types of Microtome
• ROCKING MICROTOME
Types of Microtome
• ROCKING MICROTOME
– Comprised of three moving parts
– Designed only for cutting paraffin sections
– the tissue moves through an arc as it advances
towards the knife (the slightly biconcave heiffor
knife is used) which is held rigid causing the
sections to be cut in a curved plane.
Types of Microtome
• ROCKING MICROTOME
– Very thin sections are difficult to obtain and one
major disadvantage is a limit to the size of block
which can be cut.
– Because of the lightness of the frame the microtome
has a tendency to move during cutting.
– The rocking microtome has largely been replaced
by the more precise rotary microtome although it is
re-appearing in portable cryostats. Now mainly
used for botanical applications sections of 5 µm in
thickness are possible.
Types of Microtome
• HAND MICROTOME
Types of Microtome
• HAND MICROTOME
The successful use of a hand microtome is
limited to sectioning intrinsically rigid
botanical material. It is difficult to obtain
thin, even sections of animal tissues.
Microtome Knives
• STEEL KNIVES
– manufactured from high quality carbon or tool
grade steel which is heat treated to harden the
edge.
– should be free from impurities, contain anti-
corrosives and be rust-resistant.
– The best knives are those that are fully hardened
Microtome Knives
• NON-CORROSIVE KNIVES FOR
CRYOSTATS
– manufactured from hardened, heat treated stainless
steel free from all impurities and containing 12 to
15% chromium.
Microtome Knives
• DISPOSABLE BLADES
– are essentially refined, thickened razor blades.
– produce high quality sections and have replaced
conventional microtome knives in many
instances.
– manufactured from high quality stainless steel
– edges can be coated with platinum or chromium
to enhance strength and prolong cutting life
– Teflon coated for cryostat
Advantages disposable
blades
1. Substantial cost reduction
2. Consistent section reproducibility & section quality
3. reaches cryostat chamber temperature more rapidly
than a conventional knife minimizing time delay
during blade exchanges or temperature adjustments.
4. Support of all common microtome systems
5. For all paraffin embedded samples
6. Expensive resharpening no longer required
7. No longer dependent on quality of selected
resharpening service
8. No downtime while knives are at the resharpening
service
Microtome Knives
• TUNGSTEN CARBIDE
– non corrosive
– practically non magnetic
– 100x harder than hardened tool steel
– excellent resistance to wear
– brittle because of their extreme hardness and
should be handled carefully
Microtome Knives
• GLASS KNIVES
– 'Ralph knives'
– Need Glass knife holders
– Glass knives are hard but brittle and care is
required with their handling.
– deteriorate with storage due to changes in the
'flow' or 'strain' of the glass after fracture and
from oxidation impurities remaining in the
hardened glass after manufacture. Knives should
thus be prepared immediately before use.
Microtome Knives
• GLASS KNIVES
Microtome Knives
• DIAMOND KNIVES
– manufactured from gem quality diamonds
without flaws.
– very expensive the knives are extremely
durable, because of the hardness factor of the
diamond, and are used primarily for cutting
very thin, resin sections.
– Available in different STYLES for diff.
purposes
– Resharpened by manufacturer (unless
extremely damaged)
Microtome Knives
• SAPPHIRE KNIVES
– manufactured from one piece of solid sapphire
artificially produced from an alumina
monocrystal under computer controlled
thermal conditions.
– Sapphire is harder than tungsten, carbide or
glass which ensures high durability of the
cutting edge for all types of material.
– The only restriction when using a sapphire
knife is block size as the knife edge is limited to
11 mm. A special knife holder is required.
Profile of Steel Knives
• The cutting edge of all steel knives is
produced by grinding a bevel on each side
of the knife for profiles A, B and C, or onto
the angled surface of a profile D knife. The
bevel faces enclose a sharper angle than the
main surfaces of the knife
Profile of Steel Knives
• Profile A: strongly plane concave/biconcave
– Both knives are extremely sharp and are used
for cutting soft, celloidin embedded material or
foam compounds.
– not suitable for relatively hard materials, which
cause the edge to vibrate and produce the
phenomenon known as chattering.
– To obtain the best result the knife should always
be oblique to the object when cutting sections.
Profile of Steel Knives
• Profile B: plane concave
– similar to a profile A knife but has a thicker back
– used for cutting sections from material which is
too hard to cut with a profile A knife but can also
be used for softer materials embedded in
paraffin wax.
– also suitable for cutting the softer components
(stalks, leaves) of fresh botanical specimens. This
knife should be positioned obliquely to the
material being sectioned.
Profile of Steel Knives
• Profile C: wedge Shaped / plane wedge
– has more rigidity than profile A or B knives and
can therefore be used for cutting harder materials.
– cannot be ground as sharp as profile A or B knives.
– Commonly used for cutting sections from paraffin
wax embedded material, frozen sections, cryostat
sections and for small, synthetic resin embedded
material this knife can also cut soft plastics,
rubber, wood and some textile fibres. With this
style of knife the cutting plane is transverse to the
object.
Profile of Steel Knives
• Profile D: plane Shaped
– cut hard and tough material as it has greater
stability than any of the other profile knives.
– least sharp of all of the profiles.
– commonly used for cutting synthetic resin
blocks, hard materials embedded in paraffin
wax, large wax blocks and various substances
used in industry.
Sharpening Steel Knives
• can be achieved manually or by using an
automatic knife sharpening machine.
• Automatic machines tend to remove more
metal during sharpening so that knives become
worn quickly.
• Manual methods on the other hand remove far
less metal but require more skill, experience
and time to produce a satisfactory edge.
Sharpening Steel Knives