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Microtomy
The document discusses microtomy, which is the technique of cutting extremely thin tissue sections for microscopic examination. It describes the history and development of the microtome from early manual razor cutting to modern rotary microtomes. Different types of microtomes are discussed including rotary, sliding, and freezing microtomes. Key aspects of microtomy like knife types, sharpening techniques, section cutting and processing are summarized.
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Microtomy
- 1. MICROTOM Y BYEKTA JAJODIA
- 2. At the beginningof 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
- 3. Andrew Prichard -table based model in 1835 Allowed for reduction in vibration Present day microtome : Wilhelm His, Sr. (1865) Jan Evangelista Purkyně
- 4. DEFINITION 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
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- 6. Greatest single factorin producing good sections It should be : oSharp oWell adjusted
- 7. DESIGN AND SHAPES Planar concave knives – oextremely sharp overy delicate oused with very soft samples
- 8. Wedge shaped knives– oMore stable oMost widely used oModerately hard samples oUsed for frozen & paraffin section
- 9. Chisel shaped - oblunt edge oraises stability of knife orequire more force to achieve the cut
- 10. Bi-concave knives –recommended for: Paraffin section cutting on – oRocking microtome oSledge type microtome
- 11. MATERIALS USED 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
- 12. DISPOSABLE BLADES Coatedwith Polytetraflouroethylene Mainstay in most laboratories Advantages : o Reliability of constant sharp edge o Ease to use o Low & high profiles adaptable to variety of tissue & paraffin types o Low cost
- 13. 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 planes Wedge 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
- 15. HONING Doneto restore straight cutting edge & to correct bevel Types of Hone : o Belgian Black Vein o Arkansas o Aloxite o Tam O’ Shanter Scotch Hone o Carborundum o Plate Glass
- 16. ABRASIVES AND LUBRICANTS ABRASIVES LUBRICANTS Aluminium oxide Iron oxide Silicon carbide Diamond Paraffin oil Vegetable oil Soap water
- 17. Belgian Black Vein Best & Fast Hone 1/2″ thick yellow stone backed with ½″ thick black stone Yellow side used for honing Used for coarse grinding & finishing
- 18. PLATE GLASS Used by applying an abrasive like aluminium oxide Advantage – Used for honing of all types of blades by changing the abrasive powder
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- 20. KNIFE SHARPENING MACHINE 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.
- 21. STROPPING Processof polishing an already fairly sharp edge Blunt knife cannot be sharpened on a strop Types : o Flexible/Hanging o Rigid Best strops are made from hide from the rump of horse & are marked “shell horse”
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- 23. TYPES OF MICROTOME Rotary Microtome Base Sledge Microtome Rocking Microtome Sliding Microtome Freezing Microtome Ultramicrotomes
- 24. 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)
- 25. Advantages –1. Ability to cut thin 2-3mm sections 2. 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 quality o Increased productivity o Occupational safety for technologist o Reduced incidence of repetitive motion disorders
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- 28. SLIDING MICROTOME Knife/Blade is stationary and specimen slides under it Developed for use with celoidin-embedded tissue blocks
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- 30. BASE SLEDGE MICROTOME Specimen is held stationary Knife slides across the top of the specimen Used for – oLarge blocks oHard tissue oWhole mounts oNeuropathology & ophthalmic pathology
- 31.
- 33. ROTARY ROCKING MICROTOME The name of the apparatus comes from the rocking action of the cross arm Knife is fixed Tissue block moves through an arc & strikes against the knife Used in early cryostats Disadvantages – o Size of blocks that can be cut is limited o Sections are cut in a curved plane
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- 35. Freezing microtome: oUsedfor frozen section Ultramicrotome: oUsed for electron microscopy
- 36.
- 37. PARAFFIN SECTION CUTTING Equipment required – Floatation (water bath) Slide drying oven or hot plate Fine pointed forceps Camel haired brush Scalpel, slide rack, clean slides Teasing needle Ice tray Chemical resistant pencil or pen
- 38. WATER BATH /FLOATATION BATH 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
- 39. 2-3 dropsof thymol can be added to prevent bacterial growth - this will allow the water in the bath to be used over several days
- 40. DRYING OVEN /HOT PLATE Temperature should be equal to the melting point of paraffin Lower temperature (37°C for 24 hrs ) required for - oDrying delicate tissues oTissues from the CNS Too hot oven may lead to - oDistortion of cells o Loss nuclear detail oDark pyknotic nuclei
- 41. Complete dryingoccurs in 30 mins In our automated staining machine – requires only 10 mins ( at 65 degree celcius)
- 42. BRUSH AND FORCEPS Used for removal of folds, creases and bubbles Also used for manipulating the section as it passes across the edge of the blade
- 43. 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
- 44. SECTION ADHESIVES Usedto 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 tissues oExtreme temperature oTissue containing blood and mucus o Decalcified tissues
- 45. Adhesive commonly usedare-o Mayer’s egg albumin-glycerol o Dilute serum or plasma o Gelatin in floating out bath o Starch o Poly-L-Lysine o 3-Amino Propyltriethoxysilane( APES) o Charged or Plus slides
- 46. Disadvantage of proteinadhesives - oMay give a dirty background oProne to bacterial overgrowth oProne 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
- 47.
- 48. TRIMMING OF TISSUEBLOCKS 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
- 49. 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
- 50. In difficultyduring sectioning, block face is warmed by-oWarm water oExhaled air After cutting ribbons the first section is held by forceps and the last section eased from the knife edge
- 51. FLOATING OUT SECTIONS 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 cut Done by dragging tissue paper across the surface
- 52. DRYING SECTIONS 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
- 53. CUTTING HARD TISSU ES Causes of cutting difficulty - o poor-fixation o over processing Trobleshooting - oProlonged soaking of the block in water oExposing the block surface to running tap water for 30 min oReduction in knife slant may yield result oLastly softening agents may be used
- 54. Softening agentused – oGlycerol oPhenol (5-10%)
- 55. 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 acid o5-10% Hydrochloric acid
- 56.
- 57. FROZEN AND RELATEDSECTIONS Used in – 1. Demonstrating soluble substances 2. For urgent diagnosis 3. In IF methods 4. In immunocytochemical methods 5. In diagnostic and research emzyme histochemistry , where emzymes are labile
- 58. PRINCIPLE Whentissue 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
- 59. FREEZING OF FRESHUNFIXED 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 nitrogen 3. Carbon dioxide ‘cardice’ 4. Carbon dioxide gas 5. Aerosol sprays
- 60. FIXED TISSUE ANDTHE 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
- 61. CRYOSTAT SECTIONING CABINET TEMPERATURE 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
- 62. ANTI ROLL PLATE Attached to the front of microtome Intended to stop curling of frozen sections Aligned parallel to blade edge
- 63.
- 64. 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
- 65. When placedin 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
- 66. 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
- 67. 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
- 68. PROBLEMS AND SOLUTIONFOR 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
- 69. 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
- 70. Chatter –thick & thin zone parallel to blade edge-oKnife/ block loose in holder – tighten blade levers oExcessively steep clearance angle –Reduce angle oCalcified areas in tissue – surface decalcify oOver hydration of tissue – Dehydrate oDull blade – replace/use new area of blade
- 71.
- 72. PARAFFIN SECTIONS FORMING RIBBON
- 73. Splitting ofsections at right angle to knife – oNicks in blade – replace/use different area oHard particle in tissue – if calcium- surface decal oHard particle in paraffin – remove with pointed scalpel Section do not form ribbon – oParaffin too hard – re-embed in lower melting point paraffin, warm block surface oDebris on knife edge – clean blade & its holder oClearance angle incorrect - adjust
- 74. Section attachedto block on return stroke – oInsufficient clearance angle – increase angle oDebris on blade edge – clean oDebris on block edge – trim edge of block Incomplete section – oIncomplete impregnation with paraffin – re-process block oTissue incorrectly embedded – re-embed & correct orientation oSection superficially cut – re-face block, cut deeper
- 75. Excessive compression– oDull blade - Replace oParaffin too soft – cool block face & re-cut Section expand or disintegrate on water bath – oIncomplete tissue processing – re-process oWater temp. too high – turn down the temp.
- 76. Difficulty incutting a smooth flat section – oWarm block face with warm water oGentle exhale breath onto block surface during sectioning Section roll into a tight coil instead of remain flat on knife edge – oBlade dull – replace oRake angle too small – reduce blade tilt oSection too thick – reduce thickness
- 77. CONCLUSION Practicalexperience under the guidance of a skilled tutor is required to gain confidence & co-ordination for microtomy For proper maintenance of the microtome – oFollow manufacturers instructions oImplement departmental policy
- 78. REFERENCES 1.Theory andpractice 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
- 79. T H A N K Y O U