Post on 22-Nov-2014
description
transcript
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
YOU