Microtomy is the process of cutting thin tissue sections from paraffin or frozen tissue blocks for microscopic examination.

Rotary microtome, base sledge microtome, rotary rocking microtome, sliding microtome, and ultra-microtome.

It is used to flatten tissue sections after cutting, allowing them to adhere smoothly to slides.

Disposable blades provide a sharp cutting edge, reduce artifacts, and are cost-effective compared to maintaining and sharpening steel knives.

Routine surgical material is typically cut at 3–4 µm.

Common issues include thick and thin sections, chatter, splitting, and incomplete sections. Solutions involve adjusting the clearance angle, replacing dull blades, and ensuring proper block trimming.

Section adhesives like poly-L-lysine (PLL) or 3-aminopropyltriethoxysilane (APES) are used to prevent tissue detachment during staining, especially for delicate tissues or harsh staining procedures.

Flotation bath, slide drying oven, forceps, brushes, clean slides, teasing needles, and an ice tray.

Trim the block to expose the tissue, cool it on an ice tray, and ensure the microtome is set to the correct thickness (3–4 µm).

This can result from a dull blade, incorrect clearance angle, or loose block or blade holders.

Prolonged soaking of the block or surface decalcification can help. Adjusting the knife angle may also improve results.

It dries the tissue sections on slides after floating them on the water bath, ensuring proper adhesion.

Frozen sections are used for intra-operative diagnosis, enzyme histochemistry, immunofluorescence, and immunohistochemistry.

Freeze substitution involves freezing tissue, cutting sections, and transferring them to a cold solvent (e.g., acetone) for further processing.

FIF is a technique used to demonstrate biogenic amines like catecholamines by reacting them with formalin vapor to produce fluorescent compounds.

This can be due to incorrect clearance angle, dull blades, or debris on the knife edge.

Chatter results from a loose blade or block, excessive clearance angle, or tissue that is too hard.

Use charged or adhesive-coated slides, ensure proper drying, and avoid exposure to strong alkali solutions.

Poor tissue impregnation with paraffin or water bath temperatures that are too high.

Surface decalcification can be used to soften small calcium deposits before sectioning.

Intra-operative diagnosis, Mohs surgery, sentinel node evaluation, and rapid biopsy.

Enzyme histochemistry, immunofluorescence, immunohistochemistry, and lipid/carbohydrate studies.

Frozen sections preserve labile antigens that may be destroyed by heat or fixation, making them ideal for certain immunohistochemical studies.

Freeze-drying minimizes the loss of soluble substances, displacement of cell constituents, and denaturation of proteins, making it valuable for enzyme and immunohistochemical studies.

Trimming removes excess paraffin and exposes the tissue, ensuring a flat surface for consistent sectioning.

Cooling hardens the paraffin and tissue, making it easier to cut thin, uniform sections.

Aggressive trimming or improper processing can create holes in the tissue, often due to uneven paraffin infiltration.

Fatty tissues may require lower temperatures and careful handling to prevent tearing or distortion.

A teasing needle helps manipulate tissue sections, remove folds, and position them on slides.

This can occur due to insufficient clearance angle, debris on the blade, or static electricity.

Detergent reduces surface tension, allowing tissue sections to flatten more easily on the water surface.

Use distilled water in the flotation bath and gently tease out bubbles with a brush or forceps.

The temperature should be close to the melting point of the paraffin (around 60°C) to avoid overheating and distortion.

Delicate tissues, such as those from the central nervous system, may split or crack if dried too quickly, so lower temperatures and longer drying times are needed.

Snap-freezing in isopentane cooled by liquid nitrogen is the preferred method to reduce ice crystal formation.

Fresh tissue preserves labile enzymes and antigens that may be lost during fixation or processing.

OCT (Optimal Cutting Temperature) compound acts as an embedding medium, providing support for the tissue during freezing and sectioning.

Freeze artifacts can be minimized by rapid freezing and maintaining consistent cryostat temperatures.

A static guard reduces static electricity, which can cause sections to curl or stick to the block.

Positively charged slides improve tissue adhesion, especially for sections undergoing harsh staining procedures.

Fixed tissue is harder and requires warmer cutting temperatures, while unfixed tissue is softer and sections better at colder temperatures.

Fatty tissues require colder temperatures (−20°C to −25°C) to achieve optimal consistency for sectioning.

A tape transfer system helps adhere difficult sections to slides, especially for tissues prone to cracking or rolling.

Rapid freezing preserves enzyme activity, which can be lost if the tissue is frozen too slowly.

The heat extractor removes heat generated during sectioning, maintaining a consistent temperature for the tissue block.

Clean the blade with xylene or alcohol to remove paraffin debris, and replace it if nicks or dullness are observed.

It allows precise monitoring of the cabinet and block temperatures, ensuring optimal conditions for sectioning.

Regular defrosting prevents ice buildup, which can affect the performance of the microtome and the quality of sections.

A cryostat chuck holds the tissue block in place during freezing and sectioning.

Check the thermostat, defrost the unit, and ensure the door seals are intact. Regular maintenance is key.

It allows the operator to adjust the speed of sectioning, which is critical for delicate or hard tissues.

The block face angle affects the quality of sections. An incorrect angle can cause compression or uneven sections.

It ensures the cryostat is free of contaminants, which is especially important in clinical and research settings.

Small fragments can be embedded in OCT compound or placed on a cork disk before freezing.

Gum sucrose is used to protect tissue during freezing, especially for fixed tissue sections.

Freeze-drying preserves tissue structure and minimizes the loss of soluble substances, making it ideal for enzyme and immunohistochemical studies.

Vapor fixation preserves tissue components without the need for liquid fixatives, which can alter tissue structure.

Freeze-dried tissue is extremely fragile and must be handled carefully before embedding in paraffin or resin.

Ultracryotomy produces ultra-thin sections (50–150 nm) for electron microscopy, allowing detailed study of cellular structures.

Freeze substitution preserves tissue components while replacing ice with a solvent, making it useful for enzyme localization studies.

FIF is used to demonstrate biogenic amines like catecholamines, which are important in neurological studies.

Freeze-dried sections are dry-mounted onto slides to prevent the loss of water-soluble isotopes.

Freeze-drying minimizes tissue distortion, making it ideal for SEM studies of surface structures.

Freeze-drying is time-consuming and labor-intensive, making it more suitable for research than routine clinical use.

Tissue is frozen, and the interstitial water turns to ice, acting as the embedding medium. The tissue is then sectioned in a cryostat.

Most unfixed tissues section well between −15°C and −23°C, while fixed tissues require warmer temperatures (−7°C to −12°C).

Liquid nitrogen, isopentane cooled by liquid nitrogen, dry ice, carbon dioxide gas, and aerosol sprays.

An anti-roll plate prevents frozen sections from curling during cutting by aligning parallel to the blade edge.

Slides can be coated with adhesives like gelatin-formaldehyde or poly-L-lysine to ensure tissue adherence during staining.

Freeze artifacts, tissue shattering, and difficulty in handling delicate or fatty tissues.

A cryostat is a refrigerated cabinet housing a microtome for cutting frozen tissue sections.

Regular defrosting, oiling, and decontamination are essential. Follow the manufacturer’s maintenance schedule.

Cryoembedding media like OCT compound provide support for tissue during freezing and sectioning.

They provide a sharp edge, are easy to replace, and cool quickly, making them ideal for frozen sectioning.

Ultracryotomy involves cutting ultra-thin frozen sections (50–150 nm) for electron microscopy.

Freeze-drying rapidly freezes tissue and removes water by sublimation. It is used in research for immunohistochemistry, autoradiography, and enzyme studies.