المحاضرة 6+7 - امراض نسيجية عملي

Cutting or sectioning

• In cutting Using the microtome



Traditional histological technique

  • Tissues are hardened by replacing water with paraffin.
  • The tissue is then cut in the microtome at thicknesses varying from 2 to 25 micrometers thick.
  • From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope 
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Microtome
  • A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination.
  • Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology

Types of microtomes
ماكنة دورانية  _ راوتر مدور - 1. Rotary
يحتوي على مجهر مرفق + كورسات + شاشة عرض - 2. Ultra-microtome
المايكروتوم موجود ضمن ثلاجة - 3. Freezing microtomes
شبه الخلاط نوعا ما + منشار ( ابو منشار ) - 4. Saw
5. Cryostat

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✓Manual
✓Semi-automated
✓Fully automated


Manual Rotary Microtome
• Completely manipulated by the operator

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Semi-Automated Rotary Microtome

One motor to advance either the fine or coarse hand-wheel




Fully Automated Rotary Microtome
Two motors that drive both the fine and the coarse advance hand-wheel.


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Parts of rotary microtome


كلمات واردة في الصورة اعلاه
BLOCK HOLDER
KNIFE AND ITS HOLDER
BASE (MICROTOME BODY)


Knife holder base
  • A part that anchors the knife holder to the microtome stage.
  • The knife holder base can be moved toward or away from the block, but MUST be stationary and locked during microtome.



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Knife holder

  • This part is comprised of several components including
  1. The blade clamp that holds the blade.
  2. The knife tilt for adjusting the knife angle.
  3. The face plate that guides that ribbons away from the blade and towards the operator.

Coarse handwheel
  • Moves the block holder either toward the knife or away from the knife
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Micron adjustment

Micron settings for section thickness can range from 1 to 60 microns on most microtomes.



Advancement Hand wheel
Turns in one direction and advances the block toward the knife at the specified microns.

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Safety Lock
Most hand wheels are equipped with a safety lock to prevent the wheel from releasing and having the block holder come down towards the blade while a block is inserted or removed.
It should be used anytime the operator is not sectioning paraffin blocks


Ultra Microtome
  • These are used exclusively for electron microscopy. Prepare ultrathin sections.
  • It has been reported that sections can be cut as thin as 10 nanometres.
  • 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
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Freezing microtome



  • The freezing microtome is 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.
  • To delay the thawing of sections on the knife and make it possible to transfer them directly from knife to slides. 
  • The knife is moved whilst the tissue block remains static.

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  • Used for cutting thin to semi-thin sections of fresh, frozen tissue.
  • Although other microtome can be modified for cutting frozen section, this gives the best results & is used almost universally.

Saw Microtomes
  • Saw Microtomes will cut sections from very hard material such as undecalcified bone, glass or ceramics.
  • The samples, commonly embedded in resins, are moved extremely slowly against a diamond coated saw rotating at approximately 600 rpm. 
  • Sections of 20 um or greater are possible providing the saw blade is in perfect condition.
  • Very thin sections are not possible.
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Cryostat microtome [Cryotome]



Cryostat is a refrigerated cabinet in which a specialty microtome is housed.
All the controls for the cabinet are operated outside the cabinet.

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  • Cryostat is primarily used for cutting sections of frozen tissue
  •  Frozen sections were originally produced for histological techniques, but were later used to demonstrate soluble substance and the diagnosis or urgent biopsy specimens 
  • Specimens are frozen and cut at 4-8 μm thickness in an cryo-microtome using an anti roll plate
Principle

  • When the tissue is frozen
  • the interstitial water turns into ice.
  • 👇
tissue becomes firm and acts as an embedding medium.

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Freezing agents
  • Liquefied nitrogen (-190 °C)
  • Isopentane (2-methylbutane) cooled by liquid nitrogen (-150 °C) 
  • Dry ice (-70°C)
  • Carbon dioxide gas (-70 °C)
  •  Aerosol sprays (-50°C)
Types of Knives
  • STEEL KNIVES
  • GLASS KNIVES
  • D DIAMOND KNIVES
  • SAPPHIRE KNIVES
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Disposable blades
  • Manufactured from high quality stainless steel
  • different grades according to thickness
  • The edge of disposable blades can be coated with platinum6 or chromium7 to enhance strength and prolong cutting life.


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