The ultramicrotome is a specialized instrument engineered to slice materials into sections measured in nanometers. This extreme precision is necessary because these sections are designed for study using high-resolution tools, primarily the electron microscope. By producing slices typically ranging from 20 to 150 nanometers thick, the ultramicrotome provides transparent samples that reveal the internal architecture of cells and materials.
Preparing Samples for Sectioning
Achieving a clean, ultra-thin slice requires the sample itself to be rigidly supported, as most biological tissues or soft materials are too delicate to withstand the cutting force of the knife edge. Embedding provides this structural support, where the sample is infiltrated with a liquid monomer, such as an epoxy resin. The resin is then polymerized, or hardened, around the sample to create a solid block that can be securely mounted in the microtome.
Once the sample is fully encased in the hard plastic block, the block face must be carefully trimmed to a specific shape, often a small trapezoid or pyramid frustum, with sides measuring less than a millimeter. This trimming process ensures that only a small, defined area of the sample is presented to the knife edge during the final slicing process. A small block face reduces the total cutting force and helps maintain the integrity of the section ribbon as it is produced.
The Mechanism of Ultra-Thin Slicing
The ability of an ultramicrotome to consistently slice sections only a few tens of nanometers thick relies on specialized engineering that controls the advance of the specimen block toward the stationary knife. Traditional microtomes advance the sample in micrometers, but the ultramicrotome requires a mechanism that moves the sample in increments as small as 5 to 100 nanometers between each cutting stroke. The two primary methods for this ultra-fine advance are thermal and mechanical.
The thermal advance mechanism utilizes the principle of thermal expansion to control the specimen’s movement with incredible accuracy. The sample holder is mounted on a metal rod, which is slowly heated by a low-power electrical coil, causing the rod to expand minutely toward the fixed knife edge. The precise control of the electrical current allows for a predictable and regulated rate of expansion, resulting in the required nanometer-scale advance for the next cut.
In contrast, mechanical advance systems rely on sophisticated, highly calibrated gearing or lever systems, often using a micrometer screw paired with an inclined plane. This arrangement translates a relatively large, controlled rotational movement into an extremely small linear advance of the specimen block. To prevent external influences from compromising the cut, ultramicrotomes are built with massive, robust frames and frequently employ active or passive vibration isolation systems to dampen external low-frequency noise. The entire instrument is often housed in a temperature-controlled environment to minimize thermal drift that would otherwise interfere with the programmed advance.
Specialized Cutting Tools
The extreme thinness of the sections demands a cutting edge far sharper than conventional steel blades. Ultramicrotomes utilize two main types of knives, each suited for different stages of the cutting process. Glass knives are typically made in the laboratory by controlled “balanced breaking” of thick glass strips, which creates an atomically smooth edge suitable for initial trimming and cutting semi-thin sections. These semi-thin sections (0.5 to 2 micrometers thick) are used for light microscopy analysis.
For the final, ultra-thin sections (below 150 nm), the diamond knife is the preferred tool due to its extreme hardness and durability. These knives are crafted from high-quality natural or synthetic diamonds, with the cutting edge radius sharpened to a remarkable 3 to 6 nanometers. This atomic-level sharpness is necessary to cleanly shear the hard resin and embedded sample without causing compression, tearing, or deformation of the delicate internal structures. The diamond knife is often mounted in a boat filled with water, allowing the freshly cut sections to float onto the liquid surface where they can be collected as a continuous ribbon.
Scientific Applications
The primary purpose of ultramicrotomy is the preparation of samples for Transmission Electron Microscopy (TEM). TEM utilizes a beam of electrons to create an image, and since electrons have low penetrating power, the sample must be thin enough to be electron-transparent, necessitating the nanometer thickness. Without the ultramicrotome to produce these precise 20 to 150 nanometer sections, high-resolution imaging of cellular organelles, viruses, and macromolecular complexes would be impossible.
Beyond biological research, ultramicrotomy is a valuable technique in materials science for analyzing the internal structure of polymers, ceramics, and composite materials at the nanoscale. Specialized techniques, such as cryo-ultramicrotomy, allow for sectioning samples at temperatures as low as -150°C. This method is used to section frozen, hydrated biological specimens or temperature-sensitive materials, preserving their native state without the chemical alteration or structural collapse that can occur with resin embedding. The resulting ultra-thin sections from all these methods are then placed onto a fine mesh grid for subsequent analysis.