What is a Cryostat & How Does It Work?
In clinical pathology, histology, histopathology, and research labs, it is crucial to keep tissue samples intact and provide fast results. This can be achieved using a cryostat, which uses cryogenic temperatures to freeze and preserve tissues, which can be sectioned using a microtome.
When choosing one for your lab, it’s important to make sure it is the right size, has the correct features, and can section specimens the way you need it to so that you can build the most efficient workflow possible.
What Does a Cryostat Do?
Cryostats are used to preserve frozen tissue samples, slice tissue sections thin enough for microscopic examination, and provide a quick diagnosis for a variety of diseases and medical conditions, including neuromuscular diseases. They can also be used to examine enzyme histochemistry. The entire process is performed under cryogenic temperatures.
Cryogenic temperature refers to a range of about -150℃ (-238℉) to absolute zero (-273℃ or -460℉.) This range is considered the temperature at which molecular motion comes as close as possible to stopping altogether. A cryostat maintains its temperature using a freezing chamber and cryogenic gas.
Cryostats use a number of refrigeration methods to achieve these low temperatures, including helium baths with liquid helium or liquid nitrogen. Other cryogenic fluids that may be used to cool a cryostat include argon, oxygen, and nitrogen.
How Do Cryostats Work?
A cryostat is made up of five parts that serve important functions for various experiments. These are the: freezing chamber or shelf, specimen holders, microtome, blade holder, and anti-roll guides.
Before tissues can be analyzed, they must first be prepped. Cryosectioning is best for tissues that melt, wrinkle, crack, tear, or otherwise deteriorate when you work with them. You must embed the tissue for sectioning, either in paraffin, optimal cutting temperature (OCT) compounds, or resin.
Before starting the cutting process, you can use OCT to practice to make sure your settings are correct. This way, you don’t waste valuable samples while learning to operate the machine.
The freezing shelf is a place to hold sample tissues that are stored and frozen before they are sectioned.
Freezing shelves are located near the compressor system, so they have an average working temperature -10℃ lower than the set cryostat chamber temperature. Newer cryostat units come with a Peltier freezing stage – a thermoelectric device that increases heat diffusion upon activation.
This means the unit has a higher cooling rate, which leads to a faster freezing process. Sample tissues should be small and thin, (3 to 4mm) to speed up the process and prevent ice crystals from forming.
Also called “chucks”, specimen holders mount the frozen specimen on the cryostat microtome for sectioning.
Chucks are built with a crossing grid pattern of sharply cut channels to maximize gripping power to hold the samples.
In addition, chucks are made with stainless steel so as to be able to withstand the significant freezing power, and are available in numerous shapes and sizes to accommodate multiple requirements.
The microtome is a sharp blade mounted inside the cryostat. It includes a mechanism that advances the tissue samples toward a fixed blade that sections them into microscopic pieces.
Older units come with a wheel on the outside of the chamber to manually adjust the microtomes, to help preserve the quality of the sample tissues to be sectioned. Newer units feature push-button electrical microtome controls.
The microtome allows for slicing adjustments to be made in micrometers – a unit of measurement that determines the cutting precision. It’s also possible to find machines with vibratomes, which are similar to microtomes, but use vibrating blades to cut through the tissue samples.
The blade holder is in front of the microtome. It is either fixed to the base of the microtome, or the cabinet. These clamp the cutting blade into position. Either disposable or reusable blades are used.
These blades are intended to be used for a certain amount of time before they have to be replaced.
Disposable blades have to be clamped with even pressure maintained across the entire length of the blade. If either the front or rear pressure plate sustains damage, it will change the climbing pressure which eventually negatively affects cryosectioning efficiency and quality.
The advantage is that disposable blades do not have to be resharpened, are suitable for all cryosections and applications, and are compatible with all of the common microtome systems so you don’t have to purchase different blade units for various functions.
Reusable Steel Blades
The alternative is a reusable steel blade. This rests on a support bar that’s between two pillars of a standard knife holder. Each pillar has a screw at the top to secure the blade and make sure it is clamped firmly.
These blades are made from either high-quality carbon or tool grade steel and are made with anti-corrosives. As such, they are less likely to rust and more likely to be free of impurities when compared to disposable blades. These blades do need to be sharpened on a regular basis for sectioning tissue samples.
These are used to remove sections and to avoid curling or rolling sections that are being prepared. They are made of glass plates inside an aluminum frame. The frame creates a gap between the glass slides and the upper surface of the front pressure plate so the section can slide under. Gap sizes vary and can be adjusted according to the thickness of the section.
Cryostats without anti-roll guides use the cooled brush technique to collect and gather sections. A brush pulls the section onto the pressure plate, then down the blade’s front surface as the section’s leading-edge starts to get near the blade edge.
Types of Cryostats
When deciding the type to purchase or lease, it’s crucial to know what each offers so you can make the best choice for your needs. Your choice will depend heavily on the frozen tissue you’ll be working with most often because the cutting temperature varies depending on the kind of frozen section you’re working with.
Pricing varies widely depending on the type of cryostat, the refrigeration method used, the make, model, and more. To purchase outright, expect to spend thousands of dollars on each unit, or consider leasing a cryostat machine to help you maximize your budget.
Single compressor models can control both the temperature within the freezing plate and the cooling chamber. This process typically involves an evaporator system, which allows the cooling chamber and freezing plate to reach cryogenic temperatures.
With this configuration, you cannot control the temperature of the freezing plate separately because it is integrated with the cooling chamber’s refrigeration system.
With the double compressor model, there is one compressor to control the freezing plate temperature, and another to control the cooling chamber.
Because of this, it can cool samples faster with rapid freezing and reach lower temperatures compared to a single compressor model. Compressor failure is not as likely because the compressor that’s not being used is programmed to maintain the chosen temperature, so the refrigeration isn’t interrupted.
These are cryostats that have handwheels on the side of the device. Turning the handwheel clockwise starts the sectioning process.
As the samples are brought down by the handwheel, sections are made. The trimming thickness is also carried out in the upper reversal point of the cutting motion. When using a manual sectioning cryostat, keep the rotating area of the handwheel clear and maintain a safe distance from the rotating handle.
These have a motorized cutting drive to section samples. The cutting speed can be adjusted so that harder materials can be sectioned slower than softer materials. Return cutting speed is in line with cutting speed so there is an efficient sectioning process.
Leasing vs. Buying a Cryostat & Microtome
For a fraction of the upfront cost of purchasing even new or used, you can lease cryostats. Not only does this help you stretch your budget, but it ensures you won’t be hit with unexpected repair costs since maintenance and repair is included within your lease.
Contact us today to learn more.