TEM FOR PLANTS, MICROBIAL AND ANIMAL TISSUES

PREPARATION TECHNIQUES

Transmission electron microscopy (TEM) operates under high-vacuum conditions and requires ultrathin specimens. Biological samples must therefore undergo careful processing to preserve their internal ultrastructure and enhance electron contrast.

Chemical Fixation

Samples are initially preserved using aldehyde-based fixatives, such as glutaraldehyde or formaldehyde, which cross-link proteins and stabilize cellular structures. This is commonly followed by post-fixation with osmium tetroxide, which preserves and enhances the contrast of lipid-rich membranes.

Dehydration and Embedding

Water is progressively removed from the specimen using graded concentrations of organic solvents, typically ethanol or acetone. The dehydrated tissue is then infiltrated with a liquid resin, which is subsequently polymerized to form a solid block. This enables the specimen to be cut into ultrathin sections, generally 50–100 nm thick, using an ultramicrotome.

TISSUE-SPECIFIC VARIATIONS

Although the fundamental preparation steps are similar, the structural characteristics of plant, microbial and animal samples require specific adaptations to the protocol.

Plant Tissues

Plant tissues contain thick cellulose-rich cell walls, waxy cuticles, large fluid-filled vacuoles and intercellular air spaces, all of which can hinder fixative and resin penetration. Vacuum infiltration is therefore frequently applied during fixation and resin embedding to remove trapped air and improve reagent penetration.

Microbial Samples

Microorganisms such as bacteria and fungi may be embedded in resin and ultrathin-sectioned to examine their internal ultrastructure. Small particles, isolated microorganisms and viruses can also be examined using negative staining, in which an electron-dense heavy-metal stain surrounds the specimen and produces a high-contrast outline without the need for sectioning.

Animal Tissues

Animal tissues lack rigid cell walls and are therefore generally well suited to conventional fixation, dehydration and resin-embedding procedures. After sectioning, contrasting agents are commonly applied to enhance the visualization of membranes, nuclei, cytoskeletal components and intracellular organelles.

ADVANCED AND SPECIALIZED TECHNIQUES

Specialized preparation and imaging approaches can be combined with TEM to address specific biological questions.

Immuno-TEM

Immuno-TEM uses antibodies conjugated to electron-dense markers, most commonly colloidal gold nanoparticles, to localize specific proteins, antigens or other molecular targets within cells and tissues.

Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) combines fluorescence microscopy with TEM. Fluorescently labelled cells, organelles, microorganisms or particles are first identified by light microscopy and subsequently examined at much higher spatial resolution using electron microscopy.

1. Applications

Explain the main applications of the technique:

Examination of cellular ultrastructure

Analysis of organelles, membranes, cell walls and intracellular structures

Study of host–microorganism interactions

Characterization of bacteria, fungi, viruses and biological particles

Evaluation of structural changes caused by treatments or experimental conditions

Localization of proteins using immunolabelling

Morphological characterization of biological materials, biomaterials and nanoparticles

2. Types of Samples

Clearly indicate the types of samples accepted by the service:

Plant tissues

Animal tissues

Cell cultures

Bacteria and fungi

Viruses and extracellular vesicles

Seeds, pollen and reproductive structures

Biomaterials and nanoparticles

Samples previously embedded in resin

Among others...

Please contact the CTEM technical staff before sample collection or fixation.

3. Available Preparation Procedures

In addition to conventional resin embedding, the following procedures may be included:

Primary fixation and post-fixation

Dehydration

Resin infiltration and embedding

Semithin sectioning

Ultrathin sectioning

Contrast enhancement using uranyl and lead salts

Negative staining

Immunogold labelling

Sample preparation for correlative light and electron microscopy

Preliminary light-microscopy evaluation of semithin sections

4. Semithin Sectioning

Semithin Sectioning

Before ultrathin sectioning, semithin sections are commonly obtained from resin-embedded samples. These sections are stained and examined by light microscopy to assess tissue preservation, identify regions of interest and select the most appropriate areas for subsequent TEM analysis.

5. Ultrathin Sectioning and Contrast Enhancement

Ultrathin Sectioning and Contrast Enhancement

Ultrathin sections are produced using an ultramicrotome equipped with glass or diamond knives. The sections are collected on metal grids and contrasted with electron-dense compounds, such as uranyl and lead salts, to improve the visualization of membranes, organelles and other cellular structures.

6. What Users May Receive

Depending on the services actually provided by CTEM, it may include:

Advice on experimental design

Selection and optimization of sample-preparation protocols

Resin-embedded sample blocks

Semithin and/or ultrathin sections

Grids containing ultrathin sections

TEM image acquisition

Selection of representative micrographs

High-resolution digital image files

Basic support for morphological interpretation

7. Sample Submission Requirements

Sample Submission

The quality of TEM results strongly depends on the initial collection and fixation of the specimen. Samples should be sufficiently small to allow rapid penetration of the fixative and should be processed as soon as possible after collection.

Users are strongly encouraged to contact the CTEM technical staff before collecting, fixing or transporting samples. The preparation protocol will be selected according to the sample type, experimental objective and structures of interest.

8. Limitations

A short limitations section adds scientific rigor and helps users understand the technique:

TEM requires fixed specimens and does not allow live-cell imaging.

Only a very small area of the sample is examined, so representative sampling is essential.

Immuno-TEM requires antibodies that are compatible with the selected fixation and embedding conditions.

Difficult or highly impermeable samples may require specific optimization of fixation and resin infiltration.

9. Complementary Techniques

Complementary methods available at CTEM or within the institution:

Light microscopy of semithin sections

Fluorescence microscopy

Confocal laser scanning microscopy

Correlative light and electron microscopy

Image analysis and morphometry

10. Call to Action

Planning a TEM Experiment?

Contact the CTEM technical staff before sample collection to discuss fixation, sample size, controls, embedding conditions and the most appropriate imaging strategy for your study.