Article
How to Cut Thin Sections Using Ultramicrotomy
Mastering ultramicrotomy enables researchers to unlock unparalleled detail in high-resolution imaging. This technique, vital for preparing samples for transmission electron microscopy (TEM), demands exceptional precision and discipline. By following these step-by-step guidelines, you can achieve unmatched consistency in slicing ultra-thin sections, setting a foundation for ground-breaking discoveries in microscopy.
Why Ultramicrotomy Matters
Ultramicrotomy reveals nanoscale details, transforming specimens into ultra-thin sections, often as thin as 50 nanometres, for unparalleled visualisation. Essential in materials science and life sciences, this technique unveils hidden structures critical to advancing research. Success hinges on precision, technique, and the quality of tools.
Preparing the Sample for Sectioning
A successful ultramicrotomy session begins long before the cutting stage. Preparing the sample properly ensures it can withstand the stresses of slicing while maintaining its structural integrity.
Fixation and Dehydration
Preservation of the sample’s microstructure is paramount. Fixatives like glutaraldehyde or osmium tetroxide are commonly used to stabilise biological or delicate materials. Following fixation, gradual dehydration with graded ethanol solutions removes water content without causing damage, leaving the sample ready for embedding.
Embedding
Embedding the dehydrated sample in a hard resin, such as epoxy or acrylic, provides the mechanical support required for precise sectioning. Proper polymerisation of the resin is critical—it must fully harden to avoid distortion during slicing.
Trimming
The embedded sample is then trimmed to expose a flat, smooth block face. Precision trimming tools or a sharp razor blade ensure that the face aligns perfectly with the ultramicrotome’s knife, reducing waste and enhancing section quality.
How Do You Cut Very Thin Sections?
Cutting ultra-thin sections is the core of ultramicrotomy, and achieving the right thickness and consistency is where both technique and technology come into play.
Setting Up the Ultramicrotome
The ultramicrotome is a highly specialised tool designed for precision sectioning. Begin by securely mounting the trimmed block onto the specimen holder. Ensure the block is aligned so that the knife edge will cut perpendicularly to the block face. This alignment prevents uneven sections, which can obscure critical details.
Next, adjust the ultramicrotome settings to control the section thickness. For TEM applications, the target thickness typically ranges between 50 and 100 nanometers. Small adjustments can have a significant impact, so work incrementally.
Knife Preparation
The choice and preparation of the knife are critical for success. Diamond knives, known for their durability and sharpness, are ideal for repeated use, while glass knives may be suitable for less demanding applications. Clean the knife thoroughly before use, and set it at the correct cutting and clearance angles as specified by the manufacturer.
Sectioning Technique
With the ultramicrotome and knife prepared, advance the resin block slowly toward the knife. Smooth, consistent movement is essential to produce uniform sections. As each section is cut, it will float onto the water bath attached to the knife’s edge. This bath stabilises the sections and prevents curling or tearing.
Collecting the Sections
Retrieve the floating sections using a fine loop or carefully transfer them onto TEM grids. Precision handling is essential at this stage to avoid damaging or contaminating the sections. Once transferred, they are ready for further processing or direct analysis.
Post-Sectioning Steps for Optimal Imaging
The work doesn’t end after cutting the sections. Additional steps ensure the samples are ready for their final purpose.
Staining
Thin sections are often stained with solutions like uranyl acetate or lead citrate to enhance contrast and detail. These stains interact with specific sample components, highlighting structural or compositional differences for clearer imaging.
Drying
After staining, allow the sections to dry completely before loading them into the TEM. This step is crucial to prevent artefacts during imaging, which can compromise the data collected.
Expert Tips for Better Ultramicrotomy
- Maintain Temperature Control: If your ultramicrotome includes temperature control, use it to optimise cutting conditions for different materials. Cold temperatures often improve results for soft or heat-sensitive samples.
- Watch Humidity Levels: A stable environment helps prevent section distortion. Excess humidity can cause sections to wrinkle or tear, while low humidity may lead to brittleness.
- Knife Care: Regularly clean and inspect your knife. A dull or damaged edge can ruin sections and waste valuable time.
- Practice and Patience: Mastering ultramicrotomy takes time. Experiment with settings, tools, and techniques to develop a steady hand and confidence.
Ultrathin Sectioning with CN Tech
Ultramicrotomy is essential for researchers aiming to achieve high-resolution imaging across various scientific disciplines. The precision and quality of ultra-thin sections are pivotal for accurate analysis. CN Tech offers a comprehensive range of ultramicrotomy solutions tailored to meet diverse research needs.
Our flagship product, the PowerTome, is a fully modular ultramicrotome renowned for its durability and versatility. It features a power-driven cutting stroke capable of sectioning materials from soft tissues to hard metals and ceramics, making it indispensable in both life sciences and materials research.
The LN Ultra attachment integrates seamlessly with the PowerTome for applications requiring cryo-sectioning, providing a liquid nitrogen-cooled environment ideal for producing ultra-thin cryosections. This setup is particularly beneficial for techniques like Tokuyasu immunolabelling and Cryo-Electron Microscopy of Vitreous Ice Sections (CEMOVIS).
Additionally, our ATUMtome system facilitates automated tape collection of serial sections, enabling extensive sample analysis and 3D reconstruction, which is crucial in fields such as neuroscience.
At CN Tech, our experienced team is dedicated to providing tailored solutions and support to ensure your ultramicrotomy processes yield precise and reliable results. For more information on our products and services, please visit our ultramicrotomy solutions page or contact us directly.