What is microtomy?
Microtomy is the process of cutting thin tissue sections from paraffin or frozen tissue blocks for microscopic examination.
What are the main types of microtomes?
Rotary microtome, base sledge microtome, rotary rocking microtome, sliding microtome, and ultra-microtome.
What is the purpose of a flotation (water) bath in microtomy?
It is used to flatten tissue sections after cutting, allowing them to adhere smoothly to slides.
What are the advantages of disposable blades over steel knives?
Disposable blades provide a sharp cutting edge, reduce artifacts, and are cost-effective compared to maintaining and sharpening steel knives.
What is the recommended thickness for routine paraffin sections?
Routine surgical material is typically cut at 3–4 µm.
What are common problems during paraffin sectioning, and how can they be resolved?
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.
What are section adhesives, and when are they used?
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.
What equipment is needed for paraffin sectioning?
Flotation bath, slide drying oven, forceps, brushes, clean slides, teasing needles, and an ice tray.
How do you prepare a paraffin block for sectioning?
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).
What causes thick and thin sections during microtomy?
This can result from a dull blade, incorrect clearance angle, or loose block or blade holders.
How do you handle hard tissues during paraffin sectioning?
Prolonged soaking of the block or surface decalcification can help. Adjusting the knife angle may also improve results.
What is the purpose of a slide drying oven?
It dries the tissue sections on slides after floating them on the water bath, ensuring proper adhesion.
What are frozen sections used for?
Frozen sections are used for intra-operative diagnosis, enzyme histochemistry, immunofluorescence, and immunohistochemistry.
What is freeze substitution?
Freeze substitution involves freezing tissue, cutting sections, and transferring them to a cold solvent (e.g., acetone) for further processing.
What is formaldehyde-induced fluorescence (FIF)?
FIF is a technique used to demonstrate biogenic amines like catecholamines by reacting them with formalin vapor to produce fluorescent compounds.
Why do sections curl or fail to form ribbons?
This can be due to incorrect clearance angle, dull blades, or debris on the knife edge.
What causes chatter in sections?
Chatter results from a loose blade or block, excessive clearance angle, or tissue that is too hard.
How do you prevent tissue sections from detaching during staining?
Use charged or adhesive-coated slides, ensure proper drying, and avoid exposure to strong alkali solutions.
What causes sections to expand or disintegrate on the water bath?
Poor tissue impregnation with paraffin or water bath temperatures that are too high.
How do you handle calcified tissues during sectioning?
Surface decalcification can be used to soften small calcium deposits before sectioning.
What are the clinical applications of frozen sections?
Intra-operative diagnosis, Mohs surgery, sentinel node evaluation, and rapid biopsy.
What are the research applications of frozen sections?
Enzyme histochemistry, immunofluorescence, immunohistochemistry, and lipid/carbohydrate studies.
What is the role of frozen sections in immunohistochemistry?
Frozen sections preserve labile antigens that may be destroyed by heat or fixation, making them ideal for certain immunohistochemical studies.
What is the significance of freeze-drying in research?
Freeze-drying minimizes the loss of soluble substances, displacement of cell constituents, and denaturation of proteins, making it valuable for enzyme and immunohistochemical studies.
What is the purpose of trimming a paraffin block before sectioning?
Trimming removes excess paraffin and exposes the tissue, ensuring a flat surface for consistent sectioning.
Why is it important to cool paraffin blocks before sectioning?
Cooling hardens the paraffin and tissue, making it easier to cut thin, uniform sections.
What causes “moth holes” in tissue sections?
Aggressive trimming or improper processing can create holes in the tissue, often due to uneven paraffin infiltration.
How do you handle fatty tissues during paraffin sectioning?
Fatty tissues may require lower temperatures and careful handling to prevent tearing or distortion.
What is the role of a teasing needle in microtomy?
A teasing needle helps manipulate tissue sections, remove folds, and position them on slides.
Why do sections sometimes stick to the block during the return stroke?
This can occur due to insufficient clearance angle, debris on the blade, or static electricity.
What is the purpose of adding detergent to the flotation bath?
Detergent reduces surface tension, allowing tissue sections to flatten more easily on the water surface.
How do you prevent bubbles from forming under tissue sections on slides?
Use distilled water in the flotation bath and gently tease out bubbles with a brush or forceps.
What is the recommended drying temperature for paraffin sections?
The temperature should be close to the melting point of the paraffin (around 60°C) to avoid overheating and distortion.
Why do some tissues require longer drying times?
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.
What is the best method for freezing tissue to minimize artifacts?
Snap-freezing in isopentane cooled by liquid nitrogen is the preferred method to reduce ice crystal formation.
Why is fresh tissue preferred for frozen sections?
Fresh tissue preserves labile enzymes and antigens that may be lost during fixation or processing.
What is the role of OCT compound in frozen sectioning?
OCT (Optimal Cutting Temperature) compound acts as an embedding medium, providing support for the tissue during freezing and sectioning.
How do you handle freeze artifacts in frozen sections?
Freeze artifacts can be minimized by rapid freezing and maintaining consistent cryostat temperatures.
What is the purpose of a static guard near the microtome?
A static guard reduces static electricity, which can cause sections to curl or stick to the block.
Why are positively charged slides used for frozen sections?
Positively charged slides improve tissue adhesion, especially for sections undergoing harsh staining procedures.
What is the difference between fixed and unfixed tissue in frozen sectioning?
Fixed tissue is harder and requires warmer cutting temperatures, while unfixed tissue is softer and sections better at colder temperatures.
How do you handle fatty tissues in frozen sectioning?
Fatty tissues require colder temperatures (−20°C to −25°C) to achieve optimal consistency for sectioning.
What is the purpose of a tape transfer system in frozen sectioning?
A tape transfer system helps adhere difficult sections to slides, especially for tissues prone to cracking or rolling.
Why is rapid freezing important for enzyme histochemistry?
Rapid freezing preserves enzyme activity, which can be lost if the tissue is frozen too slowly.
What is the role of the heat extractor in a cryostat?
The heat extractor removes heat generated during sectioning, maintaining a consistent temperature for the tissue block.
How do you clean and maintain a microtome blade?
Clean the blade with xylene or alcohol to remove paraffin debris, and replace it if nicks or dullness are observed.
What is the purpose of a digital temperature display in a cryostat?
It allows precise monitoring of the cabinet and block temperatures, ensuring optimal conditions for sectioning.
Why is it important to defrost a cryostat regularly?
Regular defrosting prevents ice buildup, which can affect the performance of the microtome and the quality of sections.
What is the role of a cryostat chuck?
A cryostat chuck holds the tissue block in place during freezing and sectioning.
How do you troubleshoot a cryostat that is not maintaining temperature?
Check the thermostat, defrost the unit, and ensure the door seals are intact. Regular maintenance is key.
What is the purpose of a mechanical cutting speed control in a cryostat?
It allows the operator to adjust the speed of sectioning, which is critical for delicate or hard tissues.
Why is it important to monitor the block face angle in a cryostat?
The block face angle affects the quality of sections. An incorrect angle can cause compression or uneven sections.
What is the role of a cryostat decontamination system?
It ensures the cryostat is free of contaminants, which is especially important in clinical and research settings.
How do you handle tissue fragments that are too small for a cryostat chuck?
Small fragments can be embedded in OCT compound or placed on a cork disk before freezing.
What is the purpose of gum sucrose in frozen sectioning?
Gum sucrose is used to protect tissue during freezing, especially for fixed tissue sections.
Why is freeze-drying used in research?
Freeze-drying preserves tissue structure and minimizes the loss of soluble substances, making it ideal for enzyme and immunohistochemical studies.
What is the role of vapor fixation in freeze-drying?
Vapor fixation preserves tissue components without the need for liquid fixatives, which can alter tissue structure.
How do you handle freeze-dried tissue for embedding?
Freeze-dried tissue is extremely fragile and must be handled carefully before embedding in paraffin or resin.
What is the purpose of ultracryotomy in research?
Ultracryotomy produces ultra-thin sections (50–150 nm) for electron microscopy, allowing detailed study of cellular structures.
Why is freeze substitution used in histochemistry?
Freeze substitution preserves tissue components while replacing ice with a solvent, making it useful for enzyme localization studies.
What is the role of formaldehyde-induced fluorescence (FIF) in neuropathology?
FIF is used to demonstrate biogenic amines like catecholamines, which are important in neurological studies.
How do you handle freeze-dried sections for autoradiography?
Freeze-dried sections are dry-mounted onto slides to prevent the loss of water-soluble isotopes.
What is the significance of freeze-drying in scanning electron microscopy (SEM)?
Freeze-drying minimizes tissue distortion, making it ideal for SEM studies of surface structures.
Why is freeze-drying not commonly used in clinical diagnostics?
Freeze-drying is time-consuming and labor-intensive, making it more suitable for research than routine clinical use.
What is the principle behind frozen sectioning?
Tissue is frozen, and the interstitial water turns to ice, acting as the embedding medium. The tissue is then sectioned in a cryostat.
What is the optimal temperature for cutting frozen sections?
Most unfixed tissues section well between −15°C and −23°C, while fixed tissues require warmer temperatures (−7°C to −12°C).
What are the common methods for freezing tissue?
Liquid nitrogen, isopentane cooled by liquid nitrogen, dry ice, carbon dioxide gas, and aerosol sprays.
What is an anti-roll plate, and how does it work?
An anti-roll plate prevents frozen sections from curling during cutting by aligning parallel to the blade edge.
How do you prepare slides for frozen sections?
Slides can be coated with adhesives like gelatin-formaldehyde or poly-L-lysine to ensure tissue adherence during staining.
What are the challenges of cutting frozen sections?
Freeze artifacts, tissue shattering, and difficulty in handling delicate or fatty tissues.
What is a cryostat?
A cryostat is a refrigerated cabinet housing a microtome for cutting frozen tissue sections.
How do you maintain a cryostat?
Regular defrosting, oiling, and decontamination are essential. Follow the manufacturer’s maintenance schedule.
What is the role of cryoembedding media?
Cryoembedding media like OCT compound provide support for tissue during freezing and sectioning.
What are the advantages of disposable blades in cryostats?
They provide a sharp edge, are easy to replace, and cool quickly, making them ideal for frozen sectioning.
What is ultracryotomy?
Ultracryotomy involves cutting ultra-thin frozen sections (50–150 nm) for electron microscopy.
What is freeze-drying, and what are its applications?
Freeze-drying rapidly freezes tissue and removes water by sublimation. It is used in research for immunohistochemistry, autoradiography, and enzyme studies.
