Notes on Microtome

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Notes on Microtome Page 1

NOTES IN MICROSCOPY

Microtome

An ultramicrotome used in microscopy.

A microtome (from the Greekmikros, meaning "small", and temnein, meaning "to cut") is a tool

used to cut extremely thin slices of material, known as sections. Important in science,

microtomes are used in microscopy, allowing for the preparation of samples for observationunder transmitted light or electron radiation. Microtomes use steel, glass, or diamond blades

depending upon the specimen being sliced and the desired thickness of the sections being cut.

Steel blades are used to prepare sections of animal or plant tissues for light microscopyhistology. Glass knives are used to slice sections for light microscopy and to slice very thin

sections for electron microscopy. Industrial grade diamond knives are used to slice hard

materials such as bone, teeth and plant matter for both light microscopy and for electronmicroscopy. Gem quality diamond knives are used for slicing thin sections for electron

microscopy.

Microtomy is a method for the preparation of thin sections for materials such as bones, minerals

and teeth, and an alternative to electropolishing and ion milling. Microtome sections can be made

thin enough to section a human hair across its breadth, with section thickness between 50 nm and

100 m.


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History

A diagram of a microtome drawn by Cummings in 1770.

In the beginnings of light microscope development, sections from plants and animals weremanually prepared using razor blades. It was found that to observe the structure of the specimen

under observation it was important to make clean reproducible cuts on the order of 100 m,

through which light can be transmitted. This allowed for the observation of samples using lightmicroscopes in a transmission mode.

One of the first devices for the preparation of such cuts was invented in 1770 by George Adams,Jr. (17501795) and further developed by Alexander Cummings. The device was hand operated,

and the sample held in a cylinder and sections created from the top of the sample using a hand

crank.

In 1835, Andrew Prichard developed a table based model which allowed for the vibration to be

isolated by affixing the device to the table, separating the operator from the knife.

Occasionally, attribution for the invention of the microtome is given to the anatomist WilhelmHis, Sr. (1865), In hisBeschreibung eines Mikrotoms (German forDescription of a Microtome),

Wilhelm wrote:

The apparatus has enabled a precision in work by which I can achieve sections that by hand I

cannot possibly create. Namely it has enabled the possibility of achieving unbroken sections of

objects in the course of research.

Other sources further attribute the development to a Czech physiologist Jan Evangelista Purkyn.

Several sources describe the Purkyne model as the first in practical use.


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Notes on Microtome

The obscurities in the origins of

simply cutting apparatuses, aundocumented.

At the end of the 1800s, the

microtomy, together with theallowed for the visualisation of

Today, the majority of microto

holder and an advancement memoving the sample over the k

forward such that the next cut

controlled by an adjustment mec

Applications

Microtome (C. Reichert, Vienna,

The most common applications

Traditional Histology TeThe tissue is then cut inthere the tissue can be m

dye(s) after prior remova

Cryosectioning Techniqfrozen state with a freez

examined with a light

histology (5 minutes vsachieve a quick diagnos

freezing tissue stops degr

mask its chemical compo Electron Microscopy Te

equipped with a glass o

(typically 60 to 100 na

appropriate heavy metalinstrument is often calle

glass knife or an indust

sectioning. These survey

the microtome are due to the fact that the first

nd the developmental phase of early de

development of very thin and consistently

elective staining of important cell componeicroscope details.

es are a knife-block design with a changeable

chanism. In most devices the cutting of the sife, where the advancement mechanism auto

for a chosen thickness can be made. The sec

hanism, allowing for precise control.

19051915).

f microtomes are:

chnique: tissues are hardened by replacing wa

the microtome at thicknesses varying from 2ounted on a microscope slide, stained with ap

l of the paraffin, and examined using a light mi

e: water-rich tissues are hardened by freezining microtome or microtome-cryostat; section

microscope. This technique is much faster

6 hours) and is used in conjunction with mediis. Cryosections can also be used in immuno

adation of tissue faster than using a fixative an

sition as much.chnique: after embedding tissues in epoxy rer gem grade diamond knife is used to cut v

ometer). Sections are stained with an aqueo

alt and examined with a transmission electrond an ultramicrotome. The ultramicrotome is a

rial grade diamond knife to cut survey secti

sections are generally 0.5 to 1 m thick and

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microtomes were

ices is widely

thin samples by

ts or molecules

nife, a specimen

ample begins bymatically moves

tion thickness is

er with paraffin.

to 50 m. Fromropriate aqueous

roscope.

g and cut in theare stained and

than traditional

al procedures toistochemistry as

does not alter or

in, a microtomery thin sections

s solution of an

microscope. Thislso used with its

ns prior to thin

re mounted on a


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Notes on Microtome

glass slide and stained t

sectioning for the TEM.diamond knife. Compl

increasingly found moun

be imaged and then remo

This technique is called Botanical Microtomy Te

sledge microtome. Thesregular microtome.

Spectroscopy (especiallsections are needed inexamination. It is norm

more detailed analysis o

used for sample inspectio

A recent development is the lase

laser instead of a mechanical kpreparation techniques. The las

native state. Depending on thefeasible.

Microtome types

Sledge microtome

A sledge microtome.

A sledge microtome is a device

then moves backwards and forplaced upon a linear bearing, a

sections. By adjusting the angl

applied to the sample during tmicrotome are of the preparati

biological preparations. Typical

60 m.

locate areas of interest under a light microsc

Thin sectioning for the TEM is often done wiementing traditional TEM techniques ultr

ted inside an SEM chamber so the surface of t

ved with the microtome to uncover the next sur

erial Block-Face Scanning Electron Microscopchnique: hard materials like wood, bone and

microtomes have heavier blades and cannot

FTIR or Infrared spectroscopy) Techniqu

rder that the infra-red beam will penetrate tl to cut samples to between 20 and 100 m

much smaller areas in a thin section, FTIR m

n.

r microtome, which cuts the target specimen wi

nife. This method is contact-free and does nor microtome has the ability to slice almost e

aterial being processed, slice thicknesses of 1

where the sample is placed into a fixed holde

wards across a knife. Modern sled microtomesign that allows for the microtome to readily

s between the sample and the microtome k

e cut can be reduced. Typical applications fon of large samples, such as those embedde

ut thickness achievable on a sledge microtome

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ope prior to thin

th a gem qualitymicrotomes are

e block face can

face for imaging.

(SBFSEM).leather require a

cut as thin as a

e: thin polymer

e sample underin thickness. For

icroscopy can be

th a femtosecond

t require sampleery tissue in its

0 to 100 m are

(shuttle), which

s have the sledcut many coarse

ife, the pressure

r this design ofin paraffin for

is between 1 and


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Notes on Microtome

Rotary microtome

A rotary microtome of older con

This instrument is a common mi

such that the actual cutting istypically fixed in a horizontal po

Principle of sample movement fo

In the figure to the left, the prinholder, the sample is cut by the

remains on the knife. At the higthe same thickness as the section

The flywheel in many microtomcut can be made, as the relativ

stopped during the sample cut.

truction.

crotome design. This device operates with a sta

art of the rotary motion. In a rotary microtsition.

r making a cut on a rotary microtome

ciple of the cut is explained. Through the motiknife position 1 to position 2), at which point

est point of the rotary motion, the sample holdthat is to be made, allowing for the next sectio

es can be operated by hand. This has the advaly large mass of the flywheel prevents the sa

The flywheel in newer models is often inte

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ged rotary action

me, the knife is

on of the samplethe fresh section

r is advanced byto be made.

tage that a cleanmple from being

rated inside the


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microtome casing. The typical cut thickness for a rotary microtome is between 1 and 60 m. For

hard materials, such as a sample embedded in a synthetic resin, this design of microtome canallow for good "Semi-thin" sections with a thickness of as low as 0.5 m.

Cryomicrotome

A cryomicrotome.

For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a liquidnitrogen chamber, in a so-called cryomicrotome setup. The reduced temperature allows for the

hardness of the sample to be increased, such as by undergoing a glass transition, which allows

for the preparation of semi-thin samples.[12]

However the sample temperature and the knifetemperature must be controlled in order to optimise the resultant sample thickness

Ultramicrotome

A ribbon of ultrathin sections prepared by room temperature ultramicrotomy, floating on water

in the boat of a diamond knife used to cut the sections. The knife blade is the edge at the upper

end of the trough of water.

An ultramicrotome is a main tool of ultramicrotomy. It can allow for the preparation ofextremely thin sections, with the device functioning in the same manner as a rotational

microtome, but with very tight tolerances on the mechanical construction. As a result of thecareful mechanical construction, the linear thermal expansion of the mounting is used to provide

very fine control of the thickness.

These extremely thin cuts are important for use with transmission electron microscope (TEM)

and Serial Block-Face Scanning Electron Microscopy (SBFSEM), and are sometimes also


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Notes on Microtome

region is precisely controlled,

micrometre. External to this zonthermal damage to the remainde

The laser radiation is directed o

three dimensional positioning odesired region of interest. Thescanner to cut large areas of

microdissection of internal areas

is also possible.

Microtome knives

A diamond knife blade used

transmission electron microscop

The design of a microtome knif

as well as the final sample requir

thereby limiting the interaction zone of the

the ultra-short beam application time introducof the sample.

to a fast scanning mirror based optical system

the beam crossover, whilst allowing for beacombination of high power with a high rastesample in a short time. In the laser micr

in tissues, cellular structures, and other types

or cutting ultrathin sections (typically 70

y.

is dependant upon the material and preparatio

ements (e.g. cut thickness and quality).

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cut to under a

es minimal to no

which allows for

traversal to ther rate allows thetome the laser-

of small features

to 350 nm) for

n of the samples,


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Notes on Microtome

Knife design and cut types

Profiles of microtome knives.

Generally, knives are character

categories of planar concave, we

Planar concave microtome kni

therefore only used with very so

and find use in moderately hardthe chisel profile with its bl

significantly more force to achie

For ultramicrotomes, glass and

therefore on the order of a few

microtome knives. Glass knivespecial "knife-maker" fracturi

preparations even where diamonhave small troughs, made with

float for later collection. Diamothe same collection method.

ized by the profile of the knife blade, whic

dge shaped or chisel shaped designs.

ves are extremely sharp, but are also very

ft samples. The wedge profile knives are some

materials, such as in epoxy or cryogenic samplnt edge, raises the stability of the knife,

ve the cut.

diamond knives are required, the cut breadt

illimetres and is therefore significantly smaller

are usually manufactured by the fracture ofg devices. Glass knives may be used fo

d knives may be used for final sectioning. Glaplastic tape, which are filled with water to all

d blades may be built into such an existing tro

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falls under the

delicate and are

what more stable

cutting. Finally,whilst requiring

of the blade is

than for classical

glass bars usingr initial sample

ss knives usuallyw the sample to

gh, allowing for


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Sectioning

Prior to cutting by microtomy, biological materials are usually placed in a more rigid fixative, in

a process known as embedding. This is achieved by the inflow of a liquid substance around the

sample, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to

produce a "block" which is readily cut.

The declination is the angle of contact between the sample vertical and knife blade. If the knifeblade is at right angles (declination=90) the cut is made directly using a pressure based mode,

and the forces are therefore proportionally larger. If however the knife is tilted, the relative

motion of the knife is increasingly parallel to sample motion, allowing for a slicing action. Thisbehaviour is very important for large or hard samples

The inclination of the knife is the angle between the knife face and the sample. For an optimalresult, this angle must be chosen appropriately. The optimal angle depends upon the knife

geometry, the cut speed and many other parameters. If the angle is adjusted to zero, the knife cut

can often become erratic, and a new location of the knife must be used to smooth this out.

If the angle is too large, the sample can crumple and the knife can induce periodic thickness

variations in the cut. By further increasing the angle such that it is too large one can damage theknife blade itself.

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Notes on Microtome