A cryostat is a refrigerated microtome, a specialized instrument designed to slice extremely thin sections from frozen biological specimens. This technique, known as cryosectioning, is primarily used in histology and pathology to prepare tissue for microscopic analysis, especially when rapid diagnosis is required, such as during surgical procedures. Unlike standard paraffin embedding, cryosectioning preserves sensitive molecular components like enzymes and lipids that might be destroyed by chemical fixatives or organic solvents. Maintaining a stable, low-temperature environment keeps the tissue block rigid for precision cutting.
Prepping the Equipment and Specimen
The process begins by stabilizing the cryostat’s internal chamber. The chamber temperature must be set to a specific range, typically between \(-20^{circ}text{C}\) and \(-35^{circ}text{C}\), depending on the tissue’s composition; fatty tissues, for instance, require a colder setting. This preparatory cooling period, which can take several hours, ensures that all internal components, including the microtome and the blade, are fully equilibrated to the cutting temperature.
Tissue preparation involves embedding the sample in Optimal Cutting Temperature (OCT) compound, a water-soluble blend of glycols and resins. The tissue is placed in a mold, covered with the OCT medium, and then flash-frozen, often using a quick-freeze shelf or a bath of isopentane cooled with liquid nitrogen. Rapid freezing minimizes the formation of ice crystals, which can damage cellular structure and distort tissue morphology. Once solidified, the resulting tissue-OCT block is ready for mounting onto the microtome’s specimen holder.
Mounting the Specimen and Trimming
The frozen block is secured onto a metal specimen holder, referred to as a chuck, using fresh OCT compound as an adhesive. The chuck is then mounted into the microtome’s head. Careful alignment is necessary to ensure the entire face of the tissue block is parallel to the cutting edge of the blade. This alignment is achieved using fine-adjustment knobs on the microtome head, which control the X, Y, and Z axes, reducing the amount of tissue that must be removed before the desired section is reached.
Once aligned, the initial preparatory cutting, known as trimming or roughing, begins. This involves advancing the tissue block towards the blade and making thicker cuts, typically set between 20 to 40 micrometers (\(mu\)m), to quickly plane the block face. Trimming continues until the full surface of the embedded tissue is completely exposed and a consistent face of the sample is visible. This ensures that the subsequent thin cuts capture the entire area of interest.
Precision Sectioning and Handling
After trimming, the microtome setting is switched to the final, thin-section thickness, commonly 5 to 10 \(mu\)m for microscopic viewing. Achieving consistent, high-quality sections requires a smooth and continuous rotation of the external handwheel, maintaining a steady rhythm that prevents the tissue from warming or fracturing. A slow motion can lead to compression artifacts or the section sticking to the blade, while a quick motion can cause the tissue to shatter.
To prevent the delicate, frozen section from curling as it is cut, an anti-roll plate is used. This glass or plastic device is positioned just above the blade edge. The section slides between the blade and this plate, emerging flat and ready for collection. The flattened section is gently manipulated using a fine brush or probe to remove any wrinkles before being picked up. Transfer to the slide is achieved through “thaw mounting,” where a room-temperature glass microscope slide is lightly touched to the frozen section, causing it to instantly adhere and flatten for subsequent staining or analysis.
Post-Use Care and Safety
Upon completion of the sectioning session, safety protocols must be followed, beginning with the microtome itself. The handwheel must be locked to prevent accidental movement. The disposable cutting blade must be removed with a specialized magnetic tool or forceps and immediately placed into a rigid, puncture-proof sharps container, as the exposed blade should not be handled directly.
All tissue waste, including the remaining block and trimmings, must be disposed of as biohazardous material. For cleaning, the chamber is decontaminated by wiping down all interior surfaces with gauze saturated in a laboratory-approved disinfectant, such as 70% alcohol. For a full clean, the cryostat must be completely defrosted by allowing it to reach room temperature. This facilitates the removal of frozen debris and ensures surfaces are dried before the unit is cooled down again, protecting the microtome mechanics.