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Introduction to Ultramicrotomy

Ultrathin sectioning of biological specimens and materials for microscopy and array tomography (AT)

Ultramicrotomy; ARTOS 3D Ultramicrotomy_ARTOS_3D.jpg

When studying samples, to visualize their fine structure with nanometer scale resolution, most often electron microscopy is used. There are 2 types: scanning electron microscopy (SEM) which images the sample surface or transmission electron microscopy (TEM) which requires a very thin, electron-transparent sample. Thus, to image the fine structure inside a sample using electron microscopy, the solution is to make very thin sections of it. The sample preparation method known as ultramicrotomy can produce ultrathin sections (20 to 150 nm thick) with minimal artifacts. During sectioning, the block face of the sample, from which the sections are cut, always remains a flat and straight surface which can be investigated with SEM. When the sections are imaged in an array, then a 3D image can be reconstructed of the sample. This approach is called array tomography (AT). An overview of ultramicrotomy and its use for AT is given below.

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

Array tomography (AT) is a high resolution, 3D image reconstruction method for cellular 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 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.

Principle of ultrathin sectioning

For TEM observations, as well as for optimal 3D reconstructions with array tomography, ultrathin, ordered sections are a pre-requisite. An ultramicrotome, like the UC Enuity 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.

Ultramicrotome showing the specimen arm, knife carrier, and knife with water trough.
Fig. 1: Side view of a ultramicrotome showing the specimen arm, knife carrier, and knife with water trough.

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).

Specimen preparation for array tomography

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 advanced UC Enuity 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.

Application images

Fig. 2: Image of the diamond knife of an advanced ultramicrotome solution for array tomography which performs automated serial sectioning. Wrinkle-free ribbons with uniform thickness are seen floating on the surface of water within the trough.
Fig. 2: Image of the diamond knife of an advanced ultramicrotome solution for array tomography which performs automated serial sectioning. Wrinkle-free ribbons with uniform thickness are seen floating on the surface of water within the trough.
Fig. 3: Section surface, gold – AFM image.
Fig. 3: Section surface, gold – AFM image.
Fig. 4: Cross Section, Copper and Gold Layers – TEM image. Courtesy of WWU Münster, Germany.
Fig. 4: Cross Section, Copper and Gold Layers – TEM image. Courtesy of WWU Münster, Germany.
Fig. 5: 3D image reconstruction of mouse lymph nodes acquired with array tomography. Courtesy of Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria.
Fig. 5: 3D image reconstruction of mouse lymph nodes acquired with array tomography. Courtesy of Institute of Science and Technology (IST) Austria, Klosterneuburg, Austria.
Fig. 6: Acidithiobacillus sp. HV2/2. Courtesy of Dr. Andreas Klingl, University of Marburg, Germany.
Fig. 6: Acidithiobacillus sp. HV2/2. Courtesy of Dr. Andreas Klingl, University of Marburg, Germany.
Fig. 7: Kidney, rat. Courtesy of L. Edelmann, University Hamburg, Germany.
Fig. 7: Kidney, rat. Courtesy of L. Edelmann, University Hamburg, Germany.
Fig. 8: Arabidopsis thaliana. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 8: Arabidopsis thaliana. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 9: Arabidopsis thaliana. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 9: Arabidopsis thaliana. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 10: Mouse heart. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 10: Mouse heart. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 11: Mouse heart. Courtesy of Riet De Rycke, University of Ghent, Belgium.
Fig. 11: Mouse heart. Courtesy of Riet De Rycke, University of Ghent, Belgium.

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