What is Proper Sectioning in Histology?
Proper sectioning in histology refers to the meticulous preparation of
tissue samples for microscopic examination. This involves slicing the tissue into ultra-thin sections that are typically 3-5 micrometers thick. The goal is to obtain sections that are thin enough to allow light to pass through, enabling detailed observation of the cellular structures.
Why is Proper Sectioning Important?
Proper sectioning is crucial because it directly affects the quality of the microscopic images. Poorly prepared sections can result in artifacts, distorted structures, and incomplete information, which can lead to inaccurate diagnoses. High-quality sections ensure that the
staining process is effective, revealing specific cellular components and structures with clarity.
Steps Involved in Proper Sectioning
Fixation
The first step is fixation, which involves preserving the tissue to prevent decay and maintain its structure. Common fixatives include formaldehyde and glutaraldehyde. Fixation hardens the tissue, making it easier to cut into thin sections. Embedding
After fixation, the tissue is embedded in a medium such as paraffin wax or resin. This process involves dehydrating the tissue through a series of alcohol baths, clearing it with a solvent like xylene, and then infiltrating it with the embedding medium. Once embedded, the tissue block is ready for sectioning.
Sectioning
Sectioning is performed using a microtome, a specialized instrument designed to cut ultra-thin slices of tissue. The embedded tissue block is mounted on the microtome, and a sharp blade slices it into thin sections. These sections are then floated on a water bath to remove wrinkles and transferred to glass slides for staining.
Common Problems and Solutions in Sectioning
Wrinkling
Wrinkling is a common issue that can obscure cellular details. To minimize wrinkling, ensure that the water bath is at the correct temperature and allow the sections to flatten out before transferring them to the slides. Chatter
Chatter refers to a series of parallel lines that appear on the sections due to vibrations of the microtome blade. To prevent chatter, ensure that the blade is sharp and properly aligned, and adjust the cutting speed and thickness settings on the microtome.
Compression
Compression occurs when sections appear squished or distorted. This can be caused by a dull blade or improper cutting speed. Replacing the blade and adjusting the microtome settings can help alleviate this issue.
Advanced Techniques
Cryosectioning
Cryosectioning involves freezing the tissue and cutting it with a cryostat. This technique is useful for rapid diagnosis and for tissues that are difficult to section with traditional methods. Cryosectioning preserves enzyme activity and antigenicity, making it ideal for certain types of staining and immunohistochemistry. Ultramicrotomy
For electron microscopy, ultramicrotomy is used to cut sections that are 50-100 nanometers thick. This requires an ultramicrotome and a diamond or glass knife. Ultrathin sections are essential for high-resolution imaging and detailed study of cellular organelles.
Vibratome Sectioning
A vibratome uses a vibrating blade to cut sections from fresh or fixed tissue without embedding. This method is useful for studying live tissues and for applications where traditional embedding may interfere with the analysis.
Conclusion
Proper sectioning is a fundamental skill in histology that ensures high-quality microscopic images and accurate diagnoses. By understanding the steps involved, common problems, and advanced techniques, researchers and pathologists can maximize the quality of their histological preparations, leading to better scientific and clinical outcomes.
