A cryostat is a specialized laboratory instrument important in scientific research and medical diagnostics. It is essentially a refrigerated chamber housing a microtome, a precision cutting tool. This integrated system allows for the preparation of extremely thin slices of frozen biological tissue for detailed microscopic examination. Cryostats are widely used in various fields, including pathology, histology, and molecular biology, where rapid and precise analysis of tissue samples is important.
Understanding the Cryostat
A cryostat’s primary function is to freeze and section tissue samples at very low temperatures. Freezing maintains tissue integrity and prevents degradation for accurate microscopic analysis. This low-temperature environment also prevents the formation of ice crystals that could distort the tissue’s delicate morphology.
The instrument consists of several components. The freezing chamber maintains consistent sub-zero temperatures, between -20°C and -30°C. Inside this chamber is the microtome, which includes a specimen holder (chuck) to secure the frozen tissue block, and a blade holder for the cutting blade. An anti-roll plate prevents thin tissue sections from curling, ensuring they lie flat for collection.
Safety First
Operating a cryostat involves several hazards, making strict adherence to safety protocols important. Personal protective equipment (PPE) is a primary defense, including a lab coat, eye protection (safety glasses), and disposable gloves to protect against biological hazards. Cut-resistant gloves, such as Kevlar or stainless steel mesh, are recommended for sharp blades.
Handling frozen tissue and sharp blades requires caution. Never manipulate blades with bare hands; use forceps, magnetic tools, or cut-resistant gloves for insertion, removal, or adjustment. Always keep hands clear of the blade and ensure the handwheel is locked and the blade is guarded when not actively sectioning. Tissue samples can harbor infectious agents, requiring proper handling as biohazardous material.
Proper disposal of waste is also important. Used blades must be immediately placed in a designated sharps container. Tissue trimmings and sections should be treated as biohazardous waste and discarded in appropriate biohazard bags or containers. Laboratories should have established emergency procedures, and users should know the location of first aid kits and cryostat emergency stop buttons.
Operating the Cryostat
Before beginning work, ensure the cryostat is prepared. Turn on the instrument and allow at least 30 minutes for the freezing chamber and microtome to reach the desired operating temperature (-20°C to -30°C). Ensure the cryostat chamber is clean. The handwheel should be locked, and the blade guarded before placing tools or samples inside the chamber.
To mount a sample, place the frozen tissue block onto a cryostat chuck. Use Optimal Cutting Temperature (OCT) compound to embed the tissue and secure it to the chuck. The tissue block should be oriented on the chuck for the desired sectioning plane. Once mounted, allow the OCT to freeze until opaque white, indicating it is ready for sectioning.
With the tissue block mounted, position the chuck on the microtome within the cryostat. Unlock the handwheel and advance the block towards the blade. Adjust the section thickness in micrometers for microscopic analysis; common thicknesses range from 1 to 100 microns. Engage the anti-roll plate to flatten the tissue section as it is cut and prevent curling.
During the sectioning process, rotate the handwheel smoothly to produce uniform, thin sections. As sections are cut, use a fine brush to guide them from the blade onto a pre-labeled microscope slide. The section will adhere to the warmer glass slide, often melting slightly upon contact. If sections fold or chatter, adjustments can be made (e.g., changing blade angle, anti-roll plate position, or tissue temperature). After sectioning, remove trimmed sections and debris using a large brush.
Cryostat Care and Maintenance
Routine cleaning and maintenance are important for longevity and consistent performance. After each use, remove the tissue block and clean the specimen chuck. Dispose of used blades immediately into a sharps container using appropriate tools, never bare hands.
The cryostat chamber should be defrosted regularly to prevent ice buildup, affecting temperature stability and mechanical function. This involves turning off the cooling system, allowing ice to melt, and wiping down interior surfaces. After defrosting, clean the microtome and the interior of the cryostat chamber with a suitable disinfectant, such as 70% ethanol, to remove all residue and tissue fragments. Wipe down all external surfaces. These cleaning steps prevent contamination and ensure the cryostat is ready for use.