Cryo sectioning, also known as cryosection, is a specialized laboratory technique that involves freezing biological tissue samples and then slicing them into extremely thin sections for microscopic examination. This method preserves the tissue’s delicate structural integrity and molecular components, which might be altered or lost during traditional processing. Rapidly solidifying the tissue allows for immediate analysis, making cryo sectioning a valuable tool in research and clinical settings for understanding cellular morphology and pathologies.
The Cryo Sectioning Process
The cryo sectioning process begins with the preparation of biological tissue, which is typically fresh and unfixed or briefly fixed to prevent degradation. The tissue is rapidly frozen to ensure it is firm enough for cutting and to maintain its cellular architecture. Common methods for rapid freezing include immersion in liquid nitrogen or isopentane cooled by dry ice, which helps prevent the formation of large ice crystals within the cells. The frozen tissue is then embedded in an optimal cutting temperature (OCT) compound, a gel-like substance that supports the tissue and provides a consistent matrix for sectioning. This embedded block is mounted onto a specialized holder, often referred to as a chuck.
The chuck containing the frozen tissue block is then placed inside a cryostat, which is essentially a microtome housed within a refrigerated chamber. This chamber maintains a low temperature, usually between -15°C to -30°C, with some tissues requiring colder temperatures. Within the cryostat, a precision microtome mechanism cuts the frozen tissue block into thin slices, typically 5 to 10 micrometers thick. As each section is cut, it is picked up with a fine brush or anti-roll plate and transferred onto a glass microscope slide. The sections are then ready for immediate staining or other specialized analytical techniques.
Applications and Advantages
Cryo sectioning is widely used in scientific and medical disciplines, especially when rapid tissue analysis or preservation of specific molecular components is important. In clinical diagnostics, its speed is a primary advantage, allowing pathologists to perform rapid intraoperative diagnoses during surgery. This enables surgeons to receive real-time feedback on tissue margins, helping to determine if all cancerous tissue has been removed before the operation concludes. This quick turnaround, often within minutes, is much faster than the hours or days required for traditional paraffin embedding methods.
Beyond its diagnostic utility, cryo sectioning is widely used in research fields such as molecular biology, neurobiology, and drug development. A primary advantage of this technique is its ability to preserve enzyme activity, antigens, and lipids, which can be degraded or washed away during the chemical processing of conventional paraffin embedding. This preservation is important for techniques like immunohistochemistry and immunofluorescence, where accurate detection of specific proteins and molecules is necessary. The method also supports genomic and proteomic studies by maintaining the integrity of DNA, RNA, and proteins within the tissue samples.
Overcoming Obstacles
Despite its many benefits, cryo sectioning presents several challenges that can affect sample quality and require careful technique to overcome. One common issue is the formation of ice crystals within the tissue during freezing, which can distort cellular structures and create a “Swiss cheese” appearance, known as freezing artifact. This artifact can obscure cellular details and hinder accurate diagnosis or analysis. To mitigate ice crystal formation, rapid freezing methods using cryogens like liquid nitrogen or isopentane are employed, aiming for vitrification. Pre-chilling the embedding compound and other tools within the cryostat also helps accelerate freezing and reduce artifacts.
Other artifacts that can arise during cryo sectioning include compression, tearing, and curling of the sections. Compression occurs when the tissue is squeezed by the microtome blade, distorting the section’s dimensions. Tearing can happen if the tissue is too brittle or the blade is dull, while curling makes it difficult to mount flat sections onto slides. These issues can be addressed through careful control of the cryostat’s temperature, proper maintenance and sharpness of the microtome blade, and the use of an anti-roll plate or brush to flatten sections as they are cut. Safety considerations are also important due to the use of extremely cold temperatures and sharp microtome blades, requiring appropriate personal protective equipment and careful handling.