Microtome – Principle, Parts, Types, and Uses

What is Microtome?

• A microtome is a device used for cutting extremely thin slices (called sections) of material (biological or non-biological) for microscopic examination.
• The word microtome derives from Greek mikros (small) and temnein (to cut).
• Thin sections are required so that light or electrons can pass through, making internal structure visible under microscope.
• The specimen is often embedded in a support medium (like paraffin, resin) so that stable slices may be made.
• Blades of steel, glass or diamond are used, depending on hardness of specimen.
• Sections from 1 μm to 100 nm or more thicknesses are possible in advanced instruments.
• Microscopic anatomy / histology is enabled by the use of microtome to see cell & tissue architecture.
• Diagnosis of diseases (e.g. cancer biopsies) depends on good thin sections.
• Research in developmental biology, pathology, botany, materials science is facilitated by microtomy.
• Structural details (organelles, cell walls, interfaces) are resolved because uniform slices are produced.
• Comparisons between specimens become reliable because thickness, orientation and quality are controlled.
• Before mechanical microtomes, sections were cut free-hand with razor blades, often inconsistent.
• In 1770, George Adams Jr (further developed by Alexander Cummings) made a device to cut thin wood/plant slices.
• In 1835, Andrew Prichard designed a table-based model to reduce vibration, improving section quality.
• The anatomist Wilhelm His Sr. is often credited with invention of microtome in 1865, publishing Beschreibung eines Mikrotoms.
• Jan Evangelista Purkyně and his assistants also made early prototypes (sliding microtome) in 19th century.
• Over 1800s–1900s many improvements: rocking microtomes, rotary microtomes, cryostats (1950s cryostat made by Pearse & Slee)
• Today ultramicrotomes, laser microtomes and advanced machines exist, giving ultra-thin slices for electron microscopy.

Principle of Microtome

• The principle can define as mechanical cutting of specimen into very thin, uniform sections for microscopic study.
• In this method, the specimen block is advanced slowly towards a very sharp knife, while the knife is held fixed in many types of microtome.
• Sections are produced at controlled intervals (1–10 µm, or 1–10 um) by a feed mechanism, and uniform thickness is obtained.
• The tissue, often embedded in paraffin (wax) or resin, is hardened before cutting, so tearing or distortion are reduced.
• Cutting action is performed by moving the specimen by screw/rotary feed, or by moving the knife in some designs (rotary/rocking), and precise control is given.
• The angle between specimen and knife edge is adjusted, and steady pressure, correct angle, and speed are required for clean sections.
• Ribbons of sections are formed, they are floated on a water bath and then mounted on slides for staining, this step is often done gently.
• The microtome knife may be steel, glass, or diamond depending on sample type and resolution needed, and sharpness must be maintained.
• In ultramicrotome, extremely thin slices (for TEM) are produced by use of glass or diamond knives and by very fine, controlled advancement.
• The mechanism is guided by calibrated feed (micrometer screw) so sectional thickness is reproducible, and calibration checks are required often.
• Soft tissues from explant or callus, or meristemoids and plant parts (like plumule, radicle) are processed after fixation, they are embedded and then sectioned.
• With correct embedding, internal structures (cells, nuclei, fibers) are preserved and visualised, they can be observed with less artifact, though some shrinkage may occur.
• The principle also relies on the fact that uniform mechanical advancement of specimen against a sharp edge produce slices of equal thickness, this is the main concept.
• For EM samples, sections are ultrathin and contrasted, and they are collected on grids, the process is delicate and operator skill matters a lot.
• Finally, microtome operation is taught, practiced and supervised, because consistent technique ensure reproducible results and prevent damage to sample or knife.

Parts of a microtome

Base – The base is used for giving stability and strong support to the whole instrument, it’s usually made of heavy metal so that vibration is reduced during cutting of tissue.

Knife / Blade – The knife is employed for cutting the tissue sections into thin slices, different type of blades are used depend upon sample hardness.

Knife Holder – It is used for holding the knife firmly in fixed position, by it the knife angle can adjusted for proper sectioning.

Specimen Holder (or Object Clamp) – The tissue block is placed on this part, it can be moved toward the knife by rotating handle or by automatic system.

Feed Mechanism – By this mechanism, the specimen is moved forward to the knife edge by a fixed distance after each section cut, so continuous slices are obtained.

Thickness Gauge / Scale – This part is used for setting or regulating the thickness of section, mostly measured in micrometers (µm), slight change in gauge affect uniformity of slices.

Operating Handle or Motor Drive – In manual microtome, handle is rotated for cutting, in automatic or semi-automatic ones motor drive is used for moving the specimen and knife alternately.

Stage Clamp / Block Holder – Sometimes an additional clamp is provided for extra firm holding of specimen block, specially in rotary or sledge microtome models.

Waste Tray – Thin tissue sections and debris fall on tray placed below knife to keep instrument clean during operation.

Adjustment Screws – Few screws are given for alignment and tightening of knife, holder, etc., to maintain cutting accuracy and smooth operation.

Types of Microtome

The various varieties of microtomes utilized in a conventional histology laboratory are:

Microtomes can be classified as:

1. Manual microtomes
2. Semi- automatic microtomes
3. Automatic microtomes

1. Manual Microtome – This type of microtome is operated by hand and all stages of cutting process are done manually, it is simple to use but need careful handling.

• Rocking Microtome – It has oldest design, working by rocking action due to cross arm presence. The tissue block moves through an arc toward knife (Heiffor knife) fixed firmly. Thin section comes out curved shape because of slightly biconcave knife. Though it is cheap, light-weight and reliable, vibration may occur by its light structure.
• Rotary Microtome – It is most widely used in labs. The specimen block moves up/down by rotation of handwheel, and knife remains horizontal. It gives very thin ribbons (2–3 μm). It may be semi-automatic or fully automatic by controlling block movement. But it’s expensive and not ideal for large tissue blocks, also knife faces upward which can prevail accident.
• Hand Microtome – Mostly used for rigid botanical materials only. Thin plant tissue sections can be made, but animal tissues are difficult to cut by this method.
• Vibrating Microtome – Used for unfixed / unprocessed tissues, where a vibrating knife cuts the specimen immersed in fluid to reduce tearing and heat. Speed is controlled by adjusting voltage to the blade. Often used in neurobiology and plant studies.
• Sledge Microtome – Heavy and stable instrument used for cutting large blocks. The knife slides backward–forward horizontally against fixed block. Knife is wedge-shaped (24 cm long), vibration reduced. But slow operation compared to rotary or rocking types.
• Sliding Microtome – In this type knife moves horizontally against stationary specimen placed on inclined plane. Suitable for paraffin and celloidin embedded sections.
• Freezing Microtome – Used for frozen tissues, connected with CO₂ for cooling. Knife moves and tissue remains static, each section cut advances slightly. Although easy to use, quality of thin section not always consistent.
• Cryostat Microtome – Cutting takes place in deep freeze cabinet (–10°C to –40°C). Liquid nitrogen used for cooling. Commonly applied for rapid diagnosis, enzyme histochemistry, and fluorescent antibody staining.
• Saw Microtome – A circular saw blade is used for very hard tissues like bone or teeth. Produces thicker slices (few mm) where ordinary knife cannot cut.
• Ultra Microtome – Used for ultrathin (40–100 nm) sections for Transmission Electron Microscope (TEM). Knives made of diamond/sapphire/glass. Operated by precise stepping motor and microprocessor for even cutting and reproducibility.

2. Semi-Automatic Microtome – Some steps like block movement or section thickness are motorized but knife placement and trimming done manually. It reduces operator fatigue and improves uniformity of sections.

3. Automatic Microtome – Fully automated, user just input size and direction in computer interface. It cuts thin slices with high precision and reproducibility. Used in research / diagnostic histopathology.

• Computerized Microtome – Contains thermostatic switch, cryo-scalpel, cryoplate, and semiconductor freezing system. It performs both routine paraffin and frozen sectioning (1–25 μm). Cryo-scalpel and cryoplate maintained between 0°C to –40°C.
• Laser Microtome – Used for non-contact cutting by infrared laser beam with short pulse, hence no heat damage occurs. It slices tissues precisely (5–100 μm) and suitable for sensitive biological specimens.

Types of Microtome Knife

Based on the Material Used for Construction –

1. Steel Knife – Made from high-quality carbon or tool-grade steel, these knives are hardened by heating to increase edge sharpness. The steel used must be rust-resistant; the hardness of the edge determine how finely it can cut. Commonly applied for general histological sectioning.
2. Tungsten Knife – Constructed from tungsten carbide, it’s non-magnetic and about 100× harder than steel. These knives are brittle but highly wear-resistant, capable to make up to 30,000 serial sections of undecalcified bone embedded in methacrylate before re-sharpening.
3. Diamond Knife – The blade made from gem-quality diamond, it’s very expensive but extremely durable. It’s mainly used for ultrathin sectioning in electron microscopy, giving slices as thin as few nanometers without distortion.
4. Sapphire Knife – Produced from artificial sapphire (alumina monocrystal) under computer-controlled heating. It’s harder than glass or tungsten, ensures longer life. However, it can only cut small-sized blocks (edge limit about 11 mm), hence limited in large tissue sectioning.
5. Glass Knife – Commonly used in ultramicrotomy, where thin sections (40–120 nm) are required. Known also as Ralph knife, the cutting edge is formed across glass thickness. The knife is hard yet brittle, and it should be treated before use because storage for long time reduce sharpness.
6. Disposable Knife – These are stainless-steel blades with pre-sharpened edges, used mostly in modern rotary and cryostat microtomes. A Teflon-coated surface is preferred for cryo use. They replace the old reusable steel knives and reduce maintenance work.
7. Non-Corrosive Knife – Made from heat-treated stainless steel (contains 12–15% chromium), this type used mainly in cryo-microtomes. It’s free from impurities and resistant to oxidation and corrosion caused by moisture during freezing process.

Based on the Profile / Shape of Knife –

1. Plano-concave Knife – One surface is flat while the other is concave. It’s used for cutting soft materials like celloidin-embedded tissues or foamy compounds. Knife should be placed obliquely to the object while cutting. Not suitable for hard substances due to thin fragile edge.
2. Biconcave Knife – Both surfaces are concave, though back part thicker than plano-concave type. It’s employed for cutting harder materials where stability and sharp edge required. The degree of concavity may vary among knives.
3. Wedge Knife – Has extra-thick wedge-shaped edge, more rigid than plano or biconcave knives. Used for rigid materials and for sectioning frozen or paraffin-embedded specimens. It cannot be ground to very fine sharpness, but stability during cutting is excellent. The cutting plane must placed transverse to specimen.
4. Tool Edge Knife – Designed for cutting very hard materials, has least sharpness but high stability. Edge is formed by grinding bevels on both sides of the knife surface. The face of bevel encloses sharper angle than rest of blade surfaces, improving control during sectioning.

Steps of Tissue Sectioning

1. Trimming of the Block – The paraffin block containing the embedded tissue is first trimmed, so only tissue surface remain exposed. The excess wax around block edges is removed carefully to give proper shape and flat surface for smooth sectioning.
2. Adjusting Knife and Specimen – Knife (or blade) is fixed firmly in knife holder, and its angle (usually 5°–10°) adjusted for optimal cutting. The tissue block is mounted in specimen holder with surface facing the cutting edge, alignment checked by small trial cuts.
3. Setting of Section Thickness – The thickness control knob is adjusted according to desired thickness, generally between 3–5 µm for paraffin section and 10–20 µm for frozen ones. Uneven thickness may cause tearing or compression of sections.
4. Section Cutting – The block is moved up and down (or forward–backward) toward the knife by rotating handwheel in case of rotary microtome. With each stroke, a thin slice of tissue is cut and remains attached forming continuous ribbon.
5. Ribbon Formation – The freshly cut sections form a ribbon due to adhesive nature of melted paraffin at room temperature. The ribbon is guided gently by forceps or brush to prevent folding or wrinkling.
6. Floating on Warm Water Bath – The ribbon is transferred onto a water bath kept at 40–45°C, slightly below melting point of wax. This process helps to flatten wrinkles and remove folds from tissue sections.
7. Mounting on Glass Slide – Using clean glass slide coated with adhesive (like egg albumin or Mayer’s albumin), the selected flat section is picked up carefully from water surface and placed centrally on slide.
8. Drying the Slide – Mounted slides are placed on hot plate or oven (at around 60°C) for few minutes until wax melts and section adheres firmly to slide. After drying, slides are ready for staining process.

Applications of Microtome

Some of the common uses of microtomes include:

• Used widely for preparing thin tissue sections for microscopic study / histological examination in labs.
• It applied for sectioning of animal and plant tissues, mostly after fixation and embedding process.
• In pathology labs, microtome used for diagnosis of diseases like cancer, where tissue morphology is examined.
• In research, thin slices of organs, embryos, or plant stems are prepared for study of structure and function.
• It is applied in cytology for cutting samples for cellular arrangement observation.
• Used by forensic scientists to prepare specimen slides of biological samples for crime investigations.
• In medical colleges it’s used for teaching purpose – to demonstrate micro-anatomy of different tissues.
• Microtome applied in botany to observe internal structure of plant parts (root, stem, leaf etc.).
• In zoology laboratories they used for preparing cross-sections of small organisms or organs.
• It is utilized for electron microscopy where ultrathin sections (50–100 nm) are required for high resolution imaging.
• Used by histochemists for enzyme localization and chemical composition studies within cells.
• In pharmaceutical industry, microtome used to examine tissue reaction to drugs / implants.
• Applied in paleobotany, to cut fossilized plant remains embedded in resin for structural studies.
• Microtome helps in preparing serial sections which allow 3D reconstruction of biological structures.
• In material science it sometimes used for sectioning polymer or composite materials for microscopic analysis.

Advantages of Microtome

• Very thin tissue sections can be produced by microtome for microscopic examination.
• It allow uniform and accurate section thickness, which help in better visualization of cell and tissue detail.
• By microtome, both soft and hard specimens can be sectioned easily after proper embedding (like paraffin or resin).
• Sections prepared by it are more consistent and reproducible compared with hand sectioning methods.
• The microtome operation saves time / effort when large number of tissue samples are processed.
• In pathology it is advantageous because accurate diagnosis depend on fine and clear tissue sections.
• Microtome provides smooth surface sections, which enhance staining quality and contrast during observation.
• It can be adjusted for different thickness ranges (1 µm – 100 µm) depending upon type of study or sample.
• Through use of cryostat microtome, frozen tissue can be cut without chemical fixation which preserve enzyme activity.
• The instrument can used with different blade types for various materials – biological, polymer, and plant tissues.
• Mechanical microtomes offer more safety and precision compared to manual cutting with razor blades.
• In research, it’s beneficial for serial sectioning to reconstruct 3D structure of organs or cells.
• The microtome use increase efficiency of histological laboratories by giving consistent results in lesser time.
• It reduce tissue distortion and artifact formation during cutting process, which prevail misinterpretation.
• Maintenance and calibration of microtome is easy and it can be operated by trained technician with minimum error.

Limitations of Microtome

• The microtome operation require well fixed and properly embedded tissues otherwise sections get torn or compressed.
• It can not used effectively for very soft or delicate samples without freezing or special treatment.
• The cutting process may cause artifacts, like folds, knife marks, or chatter lines, which affect microscopic observation.
• Some materials like hard bones or calcified tissues are difficult to section even after decalcification.
• Skill and experience of technician are essential because improper handling lead to uneven sections.
• Equipment need regular maintenance and calibration, otherwise accuracy of section thickness is affected.
• In paraffin method, tissue shrinkage occur due to heating or embedding, so morphology sometimes get altered.
• The knife or blade used in microtome becomes dull quickly, frequent sharpening or replacement is needed.
• Cost of automatic and cryostat microtomes are high, making them less accessible for small laboratories.
• Contamination of sample may occur during sectioning if blade or holder not cleaned properly.
• Microtome cutting speed cannot be increased much otherwise tissue distortion happen easily.
• The method is time consuming when serial sections are required for 3D reconstruction studies.
• In frozen sections, tissue cracking is common due to sudden temperature change (−20°C to room temp).
• Some biological samples like lipids or mucopolysaccharides get dissolved during processing / embedding.
• Precision depend strongly on quality of embedding medium and knife angle which vary slightly by operator.

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