Essential Guide to Ultramicrotomy

Match sample hardness and modality to knife geometry and workflow. Use dedicated diamond trim knives to create orthogonal edges; for routine RT biological ultramicrotomy in epoxy, a 45° diamond knife with a clean water boat supports stable ribbons. For polymers, ensure the knife is specified for dry or cryo cutting as needed; not all edges perform equally at cryo. Integrate ionization for static control (especially thin ribbons). Stock LR White/Lowicryl HM20 if IRF/CLEM is in scope; keep UV‑curable resin on hand to stabilize small polymer volumes. Plan collection tools (eyelash, loops with sucrose) and AT‑grade knives/boats for array tomography on wafers. Finally, schedule routine knife inspections and establish a block‑face size window (e.g., 250–1200 µm) compatible with automated alignment. Document these mappings in a one‑page SOP so core facilities and industry labs can forecast consumables and maintain reproducibility across operators.

Automated trimming and automated alignment can meaningfully improve ultramicrotomy workflows by reducing the dependence on repetitive manual adjustments and operator‑specific technique. Automation helps standardize steps that traditionally vary widely between users, which supports more consistent preparation of samples across sessions, projects, and operators. Automated trimming assists users by defining trimming steps in a structured, repeatable way. This reduces the need for continual manual correction and helps users move toward their target region more efficiently, whether they are preparing samples for transmission EM, serial sectioning, or volume EM workflows. By minimizing operator‑dependent variability, automated trimming can streamline early‑stage preparation and make the workflow easier for less‑experienced users. Automated alignment complements this by guiding users through the process of bringing the block face and knife into a suitable orientation for sectioning. Instead of relying solely on the operator’s experience to make small, iterative adjustments, automation helps reduce setup effort and can lower the learning curve for new users. This contributes to more reproducible sectioning conditions and helps maintain consistent quality over time. Together, automated trimming and automated alignment help laboratories achieve smoother setup, more predictable workflows, and sectioning quality that is less sensitive to individual technique. This consistency is particularly valuable in multi‑user or high‑throughput environments and supports more reliable results across a variety of EM applications.

Interference colors visible on the floating section provide a fast, intuitive gauge of thickness. As a rule of thumb, silver ≈ ~50 nm, shading to gold ≈ ~100 nm, with thicker sections (>100 nm) displaying progressive color shifts. Use this as a live feedback loop: set the ultramicrotome feed (e.g., 50/70/100 nm) and confirm the hue under uniform illumination; then fine‑tune cutting speed to sharpen edges and maintain ribbon stability. Because resin, illumination, and optics can influence perceived hue, establish a local calibration: collect a short series at known feed steps (e.g., 50, 70, 100 nm), image them, and build a color‑to‑thickness reference for your facility. Incorporate the color strip near the station and in training slides so new users can self‑calibrate. Establishing a consistent illumination setup helps operators judge interference colors more reliably, supporting reproducible section thickness across users.

Use material behavior and experimental goals as gatekeepers. For polymers, the primary criterion is the glass‑transition temperature (Tg): cutting below Tg preserves morphology and reduces compression; soft elastomers and unstained polyolefins often require cryo, while harder materials (e.g., PMMA) can be sectioned at room temperature. Embedding small or fragile polymer volumes in a UV‑curable resin can stabilize handling without obscuring lamellae; phase‑contrast TEM can recover detail even in low‑contrast, unstained sections. For biologicals, preserve fluorescence labels by combining HPF/FS with acrylic resins (e.g., LR White, Lowicryl HM20) and minimal heavy metal concentrations when IRF targeting is needed for CLEM; otherwise, standard epoxy + contrasting may be preferred for non‑fluorescent workflows. When the region of interest is buried deep, favor micro‑CT‑guided trimming to minimize sacrificial cuts. In all cases, pair the decision with autoalignment to ensure thin, artifact‑free ribbons regardless of modality.

Stable sections start with the environment. Aim for a low‑vibration bench or anti‑vibration table and avoid direct adjacency to centrifuges, fume‑hood blowers, or high‑traffic doors. Keep temperature stable (typically ~20–23 °C) and avoid drafts across the water boat — turbulent air alters ribbon behavior and the perceived light gap. Provide controlled, even illumination to read interference colors reliably during thickness adjustments. For electrostatic effects, integrate an ionizer near the knife/boat to stabilize ribbons and reduce “fly‑away” behavior; this is particularly helpful in dry or heated rooms and is supported in UC Enuity workflows. Finally, standardize a short pre‑cut checklist: (1) verify block‑face geometry (orthogonal edges), (2) confirm knife integrity and angle, (3) camera‑guided autoalignment (tilt/rotation/knife angle) to a uniform blue light gap, then (4) set feed/speed to your target thickness using interference color as a live readout. Taken together, these controls lower operator‑to‑operator variability and improve serial ribbon yield for array tomography and volume EM.

Ultramicrotomes offer several advantages, making them essential tools for high-resolution imaging in various scientific fields. They produce ultra-thin sections, typically between 20 and 150 nanometers thick, which allow for detailed visualization of internal structures at nanometer scale resolution. Another significant advantage is the versatility of ultramicrotomes. They can be used to prepare sections from a wide range of materials, including biological specimens, polymers, metals, and ceramics. Furthermore, the process of ultramicrotomy is relatively fast and efficient, producing clean sections with minimal artifacts. This efficiency is beneficial for researchers who need to prepare multiple samples quickly.

Cutting ultrathin sections using an ultramicrotome involves several precise steps (typical steps for room temperature applications are explained in the following). First, the sample has to be fixed, dehydrated and embedded in resin to provide mechanicyl support. Then the sample is trimmed to expose a flat, smooth block face. After mounting the trimmed block onto the ultramicrotome, the block face is sectioned perpendicularly to the knife edge. Sections are typically collected in a water boat for facilitated pick up on a TEM grid.

In ultramicrotomy, glass knifes and diamond knives are use. In particular diamond knives are commonly used due to their exceptional sharpness and durability.

• Types: There are various types of diamond knives designed for different applications, including standard ultramicrotomy, cryo ultramicrotomy (wet and dry), and material science
• Angles: Diamond knives come in different angles, typically 35°, 45°, and 55°, to suit various sectioning needs.
• Sizes: The diamond edge lengths can range from 1 to 7 mm, depending on the specific requirements

A cryo ultramicrotome is a specialized type of ultramicrotome designed to cut ultra-thin sections of specimens at very low temperatures, typically between -20°C and -150°C. It is particularly useful for preparing frozen biological specimens. Cryo ultramicrotomy helps to preserve the native structure and composition of biological samples, which otherwise can be altered by chemical fixation and dehydration.

While ultramicrotomes are invaluable for preparing ultra-thin sections for transmission electron microscopy (TEM), they do have some disadvantages:

1. Complexity: Operating an ultramicrotome requires specialized training and expertise, which can be overcome by automated approaches.
2. Time-Consuming: The preparation process of samples can be very time-consuming
3. Sample Damage: There is a risk of damaging delicate samples during sectioning.
4. Limited Sample Size: Only small samples can be sectioned, which may not be representative of the entire specimen.

Despite these challenges, the high-resolution imaging capabilities provided by ultramicrotomy make it an essential tool in many scientific fields.

The main difference between a microtome and an ultramicrotome lies in the thickness of the sections they produce and their applications: While the microtome is used to cut thin slices of biological tissues typically of a few micrometers for light microscopy, the ultramicrotome is designed to cut ultra-thin sections (50-100 nm thin) for transmission electron microscopy (TEM).

Ultramicrotomy is primarily used for preparing ultra-thin sections of specimens (50 -100 nm) for transmission electron microscopy (TEM). This technique allows scientists to visualize and analyze the internal fine structures of samples at nanometer scale resolution.