Introduction to Ultramicrotomy

Ultramicrotomy is mainly used as a sample preparation method for transmission electron microscopy (TEM). It permits the internal fine structures of samples to be visualized and analyzed at nanometer scale resolution. It produces ultrathin sections of samples in a fast and clean manner. A key advantage of ultramicrotomy is the size and homogeneity of the electron-transparent area within the sections and the speed at which the sections are produced.

Ultramicrotomy can be used for multiple types of samples, including biological specimens and industrial materials, e.g. polymers (rubber and plastics) and ductile, hard, or brittle materials (metals or ceramics). There are other techniques for preparing thin sections of these samples, like focused ion beam (FIB) milling, ion etching, tripod polishing, and electrochemical processing, but ultramicrotomy has advantages in terms of speed and cleanliness.

Array tomography (AT) is a high resolution, 3D image reconstruction method for cellular and protein structure analysis. It is performed using scanning electron microscopy (SEM) or light microscopy (LM) imaging of ordered arrays of ultrathin, resin-embedded serial sections. AT allows quantitative, volumetric structural analysis and visualization of cellular and protein structures. It has better lateral and spatial resolution than conventional confocal microscopy. In addition, a higher throughput can be achieved by partially automated examination of bio-specimens.

For TEM observations, as well as for optimal 3D reconstructions with array tomography, ultrathin, ordered sections are a pre-requisite. An ultramicrotome, like the EM UC7 from Leica Microsystems, can produce such ultrathin sample sections (20 to 150 nm thick).

To form an image of a specimen in the transmission electron microscope, electrons have to penetrate the sample without any major loss in speed. A sample’s permeability to electron radiation depends partly on its mass and thickness (thickness × density) and partly on the acceleration voltage of the electron microscope. The electrons absorbed by the specimen can cause a build-up of heat and, thus, the formation of artifacts in the object.

When sectioning with an ultramicrotome, the sample is inserted into an arm, mounted on special bearings, which performs a motorized vertical cutting movement. After the section has been cut and the specimen arm retracted, an extremely precise electromechanical feed moves the sample slightly forward by a set distance corresponding to the desired section thickness. The sectioning is performed by a vertical movement of the specimen over the extremely sharp blade of a fixed glass or diamond knife. Removing the sections directly from the knife blade is difficult, because they are so thin. They are, therefore, collected from the surface of the water bath (or with the help of a micromanipulator for the case of cryo-sectioning) after the sectioning procedure. Any further preparation can then be done that may be needed before examination with electron microscopy (EM).

To prepare soft biological specimens for AT, several steps are required. These steps include:

• Tissue fixation
• Specimen extraction and resin embedding
• Serial sectioning and section ribbon collection, forming a section array
• Staining of sections for imaging, if needed.

Then the section array is imaged with SEM or LM (often fluorescence). Afterwards, the section images from the array are merged together for 3D image reconstruction and analysis.

With many ultramicrotomes, AT sample preparation has several time-consuming and cumbersome manual steps. An advanced ultramicrotome, such as the ARTOS 3D Ultramicrotome from Leica Microsystems can speed up the preparation process by automating specimen sectioning and minimizing the time required to align the sections for SEM or LM imaging.

For more information about ultramicrotomy and array tomography, please refer to the related articles shown below.