The Principles and Practice of Cryostat Frozen Sectioning

 

I. Principles and Purpose of Frozen Sectioning

Frozen sectioning relies on low temperatures to freeze tissues, rapidly increasing their hardness to provide the necessary structural support required for sectioning. Consequently, a cryostat must continuously maintain a low-temperature operating environment.

This low-temperature freezing method offers two distinct advantages. First, it significantly shortens the workflow and time required for tissue processing. Second, it preserves the integrity of intracellular biochemical activities or antigenicity without requiring chemical fixatives, satisfying specialized requirements in both research and clinical diagnostics.

However, performing tissue sectioning via freezing demands highly skilled, well-trained, and experienced personnel. This expertise is crucial for cutting intact, thin sections and preventing tissue damage caused by low temperatures during the process. It is also worth noting that the morphological preservation of tissue sections produced by a cryostat is generally inferior to traditional paraffin sections, and issues such as distortion or deformation frequently occur.

II. The Structure and Mechanics of a Cryostat

A cryostat can be divided into two main components, featuring the core microtome unit housed inside a temperature-controlled cryochamber. While the main mechanism of the microtome is similar to a standard rotary microtome used for paraffin sectioning, its operation within a sub-zero environment requires different lubrication.

To maintain smooth movement between mechanical parts, the machine must be regularly maintained using specialized low-temperature lubricants rather than standard or room-temperature oils. Misusing regular lubricants can cause them to freeze, leading to severe mechanical failure.

Additionally, operators must wear appropriate protective gloves during use. This serves a dual purpose: it prevents frostbite from touching the freezing machine frame, and it stops moisture evaporated from the hands' sweat glands from entering the chamber. If this moisture condenses and freezes inside, it can lock up the mechanical parts or compromise the precision of the machine's operation.

The cryochamber of the cryostat should generally be kept at a standby temperature of -5°C to -10°C. Before sectioning, the temperature should be adjusted to the optimal level for the specific tissue type. Sectioning should only begin once the internal components have stabilized at this target low temperature.

Generally, it takes about 1 hour for a cryostat to cool down and stabilize for every 10°C drop. When the temperature drops below -25°C, the cooling process slows down, requiring approximately 1 hour for every 5°C decrease. The minimum temperature limit of a single-compressor cryostat varies depending on the refrigerant used, but it typically bottoms out around -30°C. When sectioning, operators must select an appropriate temperature based on the tissue characteristics and avoid running the cryostat at its absolute temperature limits.

To prevent ambient moisture from entering and condensing inside the chamber, the open time of the sliding window should be minimized to maintain a stable, low-temperature environment. In the event of a sudden power outage during operation, the machine will remain cold initially. However, once the compressor stops running and the temperature begins to rise, a large volume of water droplets will condense across the internal components.

Even if power is restored immediately, the refrigeration compressor must not be restarted right away. Doing so can cause the condensed water droplets to freeze into ice, damaging the mechanical parts. Therefore, if a cryostat is shut down, operators must ensure that all moisture inside the machine has been completely removed before restarting the cooling compressor.

Fortunately, most modern cryostats feature power-failure and defrost-protection designs. When a power outage causes the machine's internal temperature to rise above 0°C, the system automatically initiates a drying program upon reconnection to power. This program uses heat to evaporate the moisture condensed inside the chamber. It is highly recommended to let this drying cycle run for at least 72 hours to ensure the mechanical parts are completely dry, keeping the drainage port open to allow condensed water to escape.

During the heated drying cycle, the elevated temperature causes metal components to expand. It also causes the low-temperature lubricant to volatilize, deplete, or lose its lubricating functionality. Therefore, no non-essential operations should be performed while the machine is running this drying program.

Once the heated drying cycle is complete, restart the cooling chamber. After the unit reaches its standard standby temperature (-5°C to -10°C), reapply the specialized low-temperature lubricant to the relevant mechanical components for maintenance. Finally, adjust the system to the desired sectioning temperature to resume normal operations.