Transmission Electron Microscopy (TEM) at CTEM - Servicio de Microscopía Confocal y Electrónica de Transmisión de la EEZ. CSIC

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.

Ultrathin sectioning is a precision technique used to obtain electron-transparent sections of resin-embedded samples for examination by transmission electron microscopy (TEM).

The embedded specimen is trimmed to expose the region of interest and mounted in an ultramicrotome.

Ultrathin sections, typically around 50–100 nm thick, are cut using a glass or diamond knife and collected on metal grids. The sections may then be contrasted with electron-dense stains to enhance the visualization of cellular and subcellular structures.

Applications

Ultrathin sectioning is suitable for:

• Ultrastructural analysis of plant, animal and microbial samples

• Examination of cell membranes, organelles and cell walls, etc...

• Study of host–pathogen interactions

• Characterization of bacteria, fungi and intracellular microorganisms

• Analysis of biomaterials, nanoparticles and composite materials

• Immunogold labelling and immuno-TEM

• Serial sectioning and three-dimensional ultrastructural studies

• Correlative light and electron microscopy

• etc...

Sample Preparation

Before ultrathin sectioning, biological samples are usually chemically fixed, dehydrated and embedded in epoxy or acrylic resin.

The choice of fixative, embedding resin and section thickness depends on the type of specimen, the structures of interest and the intended downstream analysis.

Semithin sections are commonly examined first by light microscopy to assess sample quality and identify the most appropriate regions for ultrathin sectioning.

Contrast Enhancement

Ultrathin sections may be contrasted with electron-dense compounds, such as uranyl and lead salts, to improve the visibility of membranes, organelles and other cellular components.

The staining protocol is adapted to the type of sample, embedding resin and scientific objective.

Factors Affecting Section Quality

The quality of ultrathin sections depends on:

• Fixation and preservation of the specimen

• Resin infiltration and polymerization

• Block trimming and sample orientation

• Knife quality and cutting angle

• Cutting speed

• Section thickness

• Block hardness

• Temperature and environmental conditions

Contrast staining of grids is a fundamental step in the preparation of ultrathin sections for transmission electron microscopy (TEM). Its purpose is to increase electron contrast and facilitate the identification of membranes, organelles, cell walls, nuclear material and other structural components of the sample.

Ultrathin sections, usually embedded in resin and collected on metal grids, initially show limited contrast. To improve visualization, the sections are treated with solutions containing heavy elements with a high atomic number. These compounds scatter electrons and generate differences in image intensity.

Conventional Contrast Staining

The most common procedure involves sequential double staining with:

• Uranyl acetate, which provides general contrast and shows affinity for many cellular structures, particularly membranes, proteins and nucleic acids.

• Lead citrate, which further enhances the contrast of membranes, glycogen deposits, ribosomes and other cellular components.

The combination of uranyl acetate and lead citrate is one of the most widely used contrast-staining methods for ultrathin biological sections intended for TEM analysis.

Depending on the sample type, embedding resin and scientific objective, alternative staining agents or modified protocols may also be used.

Applications

Contrast staining of grids improves the visualization of (among others):

• Cellular and intracellular membranes

• Nuclei, chromatin and nucleoli

• Mitochondria, chloroplasts and other organelles

• Endoplasmic reticulum and Golgi apparatus

• Ribosomes and vesicles

• Plant, bacterial and fungal cell walls

• Structures associated with microorganisms

• Biomaterials, nanoparticles and hybrid structures

• Immunocytochemical and immunogold labelling

General Procedure

Grids containing ultrathin sections are placed in contact with the staining solutions for controlled periods of time. They are then carefully rinsed with high-purity water and allowed to dry before examination in the electron microscope.

Contrast staining must be carried out under clean and controlled conditions. During lead citrate staining, exposure to carbon dioxide should be minimized to prevent the formation of lead carbonate precipitates on the sections.

Factors Affecting Staining Quality

The final quality of contrast staining may depend on:

• Type and concentration of the staining agent

• Incubation time

• Section thickness

• Nature of the sample

• Type of embedding resin

• Cleanliness of the grids and laboratory materials

• Quality of the rinsing water

• pH of the staining solutions

• Presence of carbon dioxide during lead staining

• Proper filtration and storage of reagents

Overstaining may obscure fine structural details, whereas insufficient staining may produce images with poor contrast. Precipitates, contaminating particles or crystalline deposits may also interfere with observation.

Types of Grids

Sections may be collected on different types of grids depending on the application:

• Copper grids for conventional ultrastructural studies

• Nickel grids for certain immunocytochemical techniques

• Grids coated with support films for delicate samples

• Grids with different mesh sizes and geometries depending on the observation area required

The choice of grid depends on the sample type, subsequent treatment and experimental objective.

Safety

The reagents traditionally used for contrast staining contain heavy metals and must be handled, stored and disposed of in accordance with the applicable chemical safety and waste-management regulations.

Transmission Electron Microscopy (TEM) is a high-resolution imaging technique used to examine the internal ultrastructure of biological and material samples. TEM provides detailed information at the nanometre and, under suitable conditions, sub-nanometre scale.

The technique is particularly valuable for studying cellular organisation, organelles, membranes, macromolecular structures, nanoparticles and the internal structure of materials.

Principle

In TEM, a highly accelerated electron beam is transmitted through an ultrathin specimen. As electrons interact with the sample, differences in electron scattering generate image contrast.

Regions containing dense or heavy elements scatter more electrons and appear darker, whereas less dense regions allow more electrons to pass through and appear brighter.

Because electrons have a much shorter wavelength than visible light, TEM achieves substantially higher spatial resolution than conventional optical microscopy.

Main Applications

TEM is commonly used for:

• Analysis of cellular and tissue ultrastructure.

• Observation of nuclei, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus and other organelles.

• Study of biological membranes and intracellular compartments.

• Examination of plant, animal and microbial cells.

• Investigation of host–pathogen interactions.

• Characterisation of viruses, bacteria and subcellular particles.

• Analysis of nanoparticles and nanomaterials.

• Evaluation of cellular damage, degeneration and stress responses.

• Study of cell death processes, including apoptosis and autophagy.

• Examination of extracellular vesicles.

• Investigation of crystal structure, interfaces and defects in materials.

• Correlative light and electron microscopy studies.

• Immunogold localisation of specific molecules.

• Elemental analysis using energy-dispersive X-ray spectroscopy.

Biological Sample Preparation

Biological specimens require extensive preparation to preserve their ultrastructure and make them suitable for electron transmission.

Typical preparation steps include:

• Primary fixation using aldehydes such as glutaraldehyde and formaldehyde.

• Post-fixation with osmium tetroxide.

• Dehydration through a graded ethanol or acetone series.

• Infiltration and embedding in epoxy or acrylic resins.

• Ultrathin sectioning using an ultramicrotome.

• Collection of sections on metal grids.

• Contrast enhancement using heavy-metal stains such as uranyl acetate and lead citrate.

Ultrathin sections are generally between approximately 50 and 100 nm thick.

Cryogenic and Alternative Preparation

Depending on the experimental objective, TEM may also be combined with:

• Cryofixation.

• High-pressure freezing.

• Freeze substitution.

• Negative staining.

• Cryo-ultramicrotomy.

• Low-temperature embedding.

• Acrylic resins for immunolabelling.

• Minimal-staining protocols for elemental analysis.

Cryogenic approaches can improve preservation and reduce artefacts caused by chemical fixation, dehydration and resin embedding.

Immunogold Labelling

TEM can be combined with immunogold labelling to localise specific proteins, antigens or other molecules at the ultrastructural level.

Primary antibodies recognise the target molecule, while secondary antibodies coupled to colloidal gold particles provide electron-dense markers that can be visualised directly in the electron microscope.

Immunogold labelling can be applied before or after resin embedding, depending on the antigen, fixation conditions and resin type.

Advantages

• Very high spatial resolution.

• Detailed visualisation of internal cellular ultrastructure.

• Suitable for biological and material samples.

• Enables localisation of structures at nanometre scale.

• Compatible with immunogold labelling.

• Can be combined with electron diffraction and elemental analysis.

• Useful for characterising nanoparticles, organelles and membranes.

• Provides information unavailable through conventional light microscopy.

Limitations

• Requires ultrathin specimens.

• Sample preparation is time-consuming and technically demanding.

• Chemical fixation and dehydration may introduce artefacts.

• Biological samples are generally analysed under high vacuum.

• Conventional TEM does not normally allow observation of living specimens.

• The field of view is relatively small.

• Image interpretation requires specialised training.

• Quantitative conclusions require appropriate sampling and replication.

Sample Types

TEM can be applied to:

• Plant tissues.

• Animal tissues.

• Cell cultures.

• Microorganisms.

• Viruses.

• Organelles and subcellular fractions.

• Extracellular vesicles.

• Nanoparticles.

• Polymers and composite materials.

• Crystalline and amorphous materials.

• Ultrathin resin sections.

• Negatively stained suspensions.

Sample Preparation Considerations

Reliable TEM analysis requires careful optimisation of:

• Fixation conditions.

• Sample size.

• Buffer composition.

• Dehydration procedure.

• Resin type.

• Polymerisation conditions.

• Section thickness.

• Grid material.

• Contrast staining.

• Antigen preservation for immunolabelling.

• Prevention of contamination, extraction and structural distortion.

Appropriate controls and representative sampling are essential, particularly when comparing experimental treatments or performing quantitative ultrastructural analysis.

Transmission Electron Microscopy provides detailed structural and compositional information at the nanoscale and is an essential technique for investigating cellular ultrastructure, biological organisation and material properties.