MICROTOMY

BY EKTA JAJODIA

At the beginning of light microscopy sections were manually prepared using razor blades.

1st devices invented - George Adams, Jr. 1770 Developed by Alexander Cummings

 The device was hand operated with the sample held in a cylinder

 Andrew Prichard - table based model in 1835

Allowed for reduction in vibration

Present day microtome :

Wilhelm His, Sr. (1865)

 Jan Evangelista Purkyně

Microtomy – means by which tissue can be sectioned and attached to a surface for further microscopy

Microtome - oMikros - "small“oTemnein - "to cut“oSectioning instrument for cutting extremely thin slices of tissues

DEFINITION

MICROTOME KNIVES

Greatest single factor in producing good sections

It should be :oSharpoWell adjusted

Planar concave knives – oextremely sharpovery delicateoused with very soft samples

DESIGN AND SHAPES

 Wedge shaped knives –oMore stableoMost widely usedoModerately hard samplesoUsed for frozen & paraffin section

Chisel shaped - oblunt edge oraises stability of knifeorequire more force to achieve the cut

Bi-concave knives – recommended for:Paraffin section cutting on –oRocking microtomeoSledge type microtome

Steel blades - prepare sections for light microscopy & frozen sections

Glass blades - for light microscopy and semi-thin sections for electron microscopy

Diamond blades - for hard materials like bone, teeth for both light & electron microscopy

Disposable blades – for routine microtomy & cryotomy

MATERIALS USED

DISPOSABLE BLADESCoated with Polytetraflouroethylene

Mainstay in most laboratories

Advantages : o Reliability of constant sharp edgeo Ease to useo Low & high profiles adaptable to variety of tissue & paraffin typeso Low cost

ANGLES OF MICROTOMY KNIVES Cutting edge (radius of curvature - 0.1 – 0.35 μm) – is the straight line formed by intersection of 2 planes, the cutting facets (length – 0.1- 0.6 mm) Bevel angle ( 27⁰ - 32⁰) – angle between the planesWedge angle ( 15⁰) – angle between the sides of the knives Clearance angle (5⁰-10⁰)- angle between cutting facet and block of tissue Rake angle - angle between upper surface of the cutting facet and the surface of the block

HONING Done to restore straight cutting edge & to correct bevel

Types of Hone :

o Belgian Black Vein o Arkansaso Aloxiteo Tam O’ Shanter Scotch Honeo Carborundumo Plate Glass

ABRASIVES AND LUBRICANTS

Aluminium oxide Iron oxide Silicon carbide Diamond

Paraffin oil Vegetable oil Soap water

ABRASIVES LUBRICANTS

Best & Fast Hone

1/2″ thick yellow stone backed with ½″ thick black stone

Yellow side used for honing

Used for coarse grinding & finishing

Belgian Black Vein

Used by applying an abrasive like aluminium oxide

Advantage – Used for honing of all types of blades by changing the abrasive powder

PLATE GLASS

TECHNIQUE OF HONING

For quick honing within 10 mins Unskilled junior can also obtain good finish Employs revolving cast iron/glass wheel Lapping compound like alumina(coarse/fine), suspension fluid & water used Proper cleaning & maintenance of the machine is necessary Fully automatic knife sharpener – employs high frequency vibrating hone plate.

KNIFE SHARPENING MACHINE

Process of polishing an already fairly sharp edge Blunt knife cannot be sharpened on a strop Types :o Flexible/Hangingo Rigid

Best strops are made from hide from the rump of horse & are marked “shell horse”

STROPPING

TECHNIQUE OF STROPPING

Rotary MicrotomeBase Sledge MicrotomeRocking MicrotomeSliding MicrotomeFreezing MicrotomeUltramicrotomes

TYPES OF MICROTOME

ROTARY MICROTOME

Referred to as “MINOT” after its inventor Mechanism – rotation of a fine advance hand wheel through 360⁰ moving the specimen vertically past the cutting surface & returning to original position

It may be – 1.Manual (completely manipulated by

operator)2.Semi-automated (one motor)3.Fully automated (two motors)

Advantages – 1. Ability to cut thin 2-3mm sections2. Easy adaptation to all types of tissue sectioning (hard, fragile, fatty)3. Ideal for cutting serial sections

Disadvantage – Manual ones can cause repetitive motion disorder

Advantages of automated rotary microtome – o Improved section qualityo Increased productivityo Occupational safety for technologisto Reduced incidence of repetitive motion disorders

ROTARY MICROTOME

SLIDING MICROTOME

Knife/Blade is stationary and specimen slides under it

Developed for use with celoidin-embedded tissue blocks

SLIDING MICROTOME

Specimen is held stationaryKnife slides across the top of the specimen

Used for – oLarge blocksoHard tissueoWhole mountsoNeuropathology & ophthalmic pathology

BASE SLEDGE MICROTOME

BASE SLEDGE MICROTOME

ROTARY ROCKING MICROTOME

The name of the apparatus comes from the rocking action of the cross arm

Knife is fixedTissue block moves through an arc & strikes against the knife

Used in early cryostats

Disadvantages – o Size of blocks that can be cut is limitedo Sections are cut in a curved plane

ROTARY ROCKING MICROTOME

Freezing microtome:oUsed for frozen section

Ultramicrotome:oUsed for electron microscopy

Freezing microtome

Ultramicrotome

Equipment required –

Floatation (water bath) Slide drying oven or hot plateFine pointed forcepsCamel haired brush Scalpel, slide rack, clean slidesTeasing needle Ice tray Chemical resistant pencil or pen

PARAFFIN SECTION CUTTING

A thermostatically controlled water bath used for floating out tissue ribbons after sectioning

Temperature of water bath - 10°C below the melting point of the paraffin

To prevent water bubbles trapped under the section distilled water is used (tap water gives rise to air bubbles)

Alcohol or a small drop of detergent may be added to water to reduce the surface tension

WATER BATH / FLOATATION BATH

2-3 drops of thymol can be added to prevent bacterial growth - this will allow the water in the bath to be used over several days

Temperature should be equal to the melting point of paraffin

Lower temperature (37°C for 24 hrs ) required for -oDrying delicate tissuesoTissues from the CNS

Too hot oven may lead to -oDistortion of cellso Loss nuclear detail oDark pyknotic nuclei

DRYING OVEN / HOT PLATE

Complete drying occurs in 30 mins

In our automated staining machine – requires only 10 mins ( at 65 degree celcius)

Used for removal of folds, creases and bubbles

Also used for manipulating the section as it passes across the edge of the blade

BRUSH AND FORCEPS

SLIDES (76*25) mm are universally used thickness – 1-1.2mm

Slides with ground and polished edges should be used to avoid danger of finger cuts

Identification details are made on slide by diamond markers

Preferred is frost-ended slides – identification mark inscribed using a soft pencil

Used to prevent section detachment from slide during staining

Causes of section detachment –oExposure to strong alkali solution during staining oCryostat section for immunofluorescence, immunohistochemistry or intraoperative diagnosis oCNS tissuesoExtreme temperature oTissue containing blood and mucus o Decalcified tissues

SECTION ADHESIVES

Adhesive commonly used are-o Mayer’s egg albumin-glycerolo Dilute serum or plasmao Gelatin in floating out batho Starch o Poly-L-Lysineo 3-Amino Propyltriethoxysilane( APES)o Charged or Plus slides

Disadvantage of protein adhesives -oMay give a dirty backgroundoProne to bacterial overgrowthoProne to heavy staining

2 adhesives are favoured –1. Poly-L-lysine – we use for IHC, IF and

cryostat 2. 3-APES

For routine HPE – we use Meyer’s egg albumin

Practical microtomy

TRIMMING OF TISSUE BLOCKS

An old knife/blade may be used

Trimming done at 15microm thickness for almost all tissues

For small biopsies like endoscopic biopsies and core biopsies of renal or liver – 10 microm

Aggressive trimming will cause moth-hole artifact

After trimming – block is placed on ice for some minutes

ADVANTAGES of this – 1. Cooling both tissue and wax and giving them a similar consistency 2. A small amount of water gets absorbed into the tissue, slightly swelling it and making sectioning easier

Routine sections are cut at 3-4 μm

Serial sections are best cut in ribbons of 10-12 inches in length, successive section sticking edge-to-edge

In difficulty during sectioning, block face is warmed by- oWarm wateroExhaled air

After cutting ribbons the first section is held by forceps and the last section eased from the knife edge

FLOATING OUT SEC TIONS Sections are floated in the water bath to remove folds Ideal floatation time is 30 seconds Prolonged floatation causes tissue expansion & distortion

Pre-flooded slides with 50% alcohol – used for circular structure like eye

Water bath to be cleaned to remove debris after each cutDone by dragging tissue paper across the surface

Small amount of water held under the section will allow further flattening to occur when heat is applied to dry the section

Temperature should be at the melting point of the paraffin For delicate tissues the temperature is reduced and time is prolonged

DRYING SECTIONS

Causes of cutting difficulty -o poor-fixationo over processing

Trobleshooting - oProlonged soaking of the block in wateroExposing the block surface to running tap water for 30 minoReduction in knife slant may yield result oLastly softening agents may be used

CUTTING HARD TISSU ES

Softening agent used – oGlyceroloPhenol (5-10%)

SURFACE DECALCIFICATION Placing the block face down in a dish that contains a small amount of decalcification solution

The block is rinsed well and blotted dry Immediate section taken since decal achieved is limited In over decalcification – diagnostic material may be compromised Common decalcification solutions –o5-10% Nitric acido5-10% Hydrochloric acid

FROZEN SECTIONS

FROZEN AND RELATED SECTIONS

Used in –1. Demonstrating soluble substances2. For urgent diagnosis3. In IF methods4. In immunocytochemical methods5. In diagnostic and research emzyme

histochemistry , where emzymes are labile

PRINCIPLE

When tissue is frozen , water within the tissue turns to ice, and in this state tissue is firm, the ice acting as the embedding medium

THE CRYOSTAT

Refrigerated cabinet in which a modified microtome is housed (usually a rotary type) Sections are best produced from fresh unfixed tissue

FREEZING OF FRESH UNFIXED TISSUE

Freezing should be as rapid as possible Slow freezing cause distortion of tissue due to ice crystal artifact freezing techniques are –1. Liquified nitrogen (-190°C )2. Isopentane cooled by liquid nitrogen3. Carbon dioxide ‘cardice’4. Carbon dioxide gas5. Aerosol sprays

FIXED TISSUE AND THE CRYOSTAT

Tissue must be absolutely fresh

Placed in formol-calcium at 4°C for 18 hrs and then slowly frozen

No diagnostic importance

To obtain accurate localisation of hydrolytic enzymes and some antigens

CRYOSTAT SECTIONING

Most unfixed tissue will section well between -15° to -23°C Fixed tissue at -7° to -12° C Tissue containing large amount of water section best at warmer temperature If contains large amount of fats – requires a very cold temperature If chatter lines present – indication that block is too cold

CABINET TEMPERATURE

ANTI ROLL PLATE

Attached to the front of microtome Intended to stop curling of frozen sections Aligned parallel to blade edge

ULTRAMICROTOMY

GLASS KNIVES Prepared from commercially available plate glass strips measuring (40*2.5) cm

3 diff thickness is available – 6.4, 8 and 10 mm

2 triangular shaped knives are produced with different cutting edge angles are produced

Higher angle knives (upto 55°) – best for cutting hard materials

Shallow angles (35°) – for softer blocks

When placed in ultramicrotome and viewed under the microscope – cutting edge appears as bright line against a dark b/g

The left 1/3rd of cutting edge s/b smooth and used for cutting thin sections

The middle third – best for trimming and cutting semi-thin sections

A trough is attached to the knife for floating out thin sections after they are cut

DIAMOND KNIVES

Already mounted in a metal block (incorporating a section collecting trough)

Cutting edge must be clean – polystyrene strips are available for this purpose

TROUGH FLUIDS

Fluid used is distilled water

10-15% solutions of ethanol or acetone may be used It is important to insure the correct level of fluid is added

Level can be seen under the ultramicrotome microscope as a flat, silvery-gold reflection which should reach right to the knife edge

Fluid can be added or removed from trough using a fine pipette

PROBLEMS AND SOLUTION FOR PARAFFIN SECTION

Ribbon/consecutive section curved –

oBlock edge not parallel – trim the block until parallel

oDull blade edge – replace blade

oExcessive paraffin – trim away excess paraffin

Thin & thick sections –

o Paraffin too soft for tissue/condition – remove excess paraffin from blade edge

o Insufficient clearance angle – increase clearance angle

o Faulty microtome mechanism – maintain microtome

o Blade/block loose in holder - tighten block & blade

Chatter – thick & thin zone parallel to blade edge-

oKnife/block loose in holder – tighten blade leversoExcessively steep clearance angle –Reduce angleoCalcified areas in tissue – surface decalcifyoOver hydration of tissue – Dehydrate oDull blade – replace/use new area of blade

CHATTER EFFECT

PARAFFIN SECTIONS FORMING RIBBON

Splitting of sections at right angle to knife – oNicks in blade – replace/use different areaoHard particle in tissue – if calcium- surface decaloHard particle in paraffin – remove with pointed scalpel

Section do not form ribbon – oParaffin too hard – re-embed in lower melting point paraffin, warm block surfaceoDebris on knife edge – clean blade & its holderoClearance angle incorrect - adjust

Section attached to block on return stroke – oInsufficient clearance angle – increase angleoDebris on blade edge – cleanoDebris on block edge – trim edge of block

Incomplete section – oIncomplete impregnation with paraffin – re-process blockoTissue incorrectly embedded – re-embed & correct orientationoSection superficially cut – re-face block, cut deeper

Excessive compression – oDull blade - ReplaceoParaffin too soft – cool block face & re-cut

Section expand or disintegrate on water bath – oIncomplete tissue processing – re-processoWater temp. too high – turn down the temp.

Difficulty in cutting a smooth flat section –oWarm block face with warm wateroGentle exhale breath onto block surface during sectioning

Section roll into a tight coil instead of remain flat on knife edge – oBlade dull – replaceoRake angle too small – reduce blade tiltoSection too thick – reduce thickness

CONCLUSION Practical experience under the guidance of a skilled tutor is required to gain confidence & co-ordination for microtomy

For proper maintenance of the microtome – oFollow manufacturers instructionsoImplement departmental policy

1.Theory and practice of histological techniques by John D Bancroft and Marilyn Gamble

2. Handbook of histopathological and histochemical techniques by C. F. A. Culling

3. Internet sources

REFERENCES

THANK

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