Brightfield Microscopy (BFM) at CTEM - Servicio de Microscopía Confocal y Electrónica de Transmisión de la EEZ. CSIC

Microtome sectioning is used to obtain thin sections of biological samples for examination by light microscopy. Depending on the specimen and the experimental objective, tissues may be embedded in paraffin, resin or another suitable support medium before sectioning.

The embedded sample is mounted in the microtome and cut using a precision blade. The resulting sections are collected on glass slides and may subsequently be stained, labelled or processed for histological, immunohistochemical, fluorescence or morphometric analysis.

Applications

Microtome sectioning is suitable for (among others):

• Histological examination of plant and animal tissues

• Analysis of tissue organization and cellular morphology

• Paraffin-embedded tissue sectioning

• Resin-embedded semithin sectioning

• Immunohistochemistry and immunofluorescence

• In situ hybridization

• Morphometric and quantitative image analysis

• Identification of regions of interest for subsequent electron microscopy

• Evaluation of structural alterations caused by treatments or experimental conditions

Sample Preparation

The sample-preparation protocol depends on the type of tissue, the embedding medium and the downstream application. Samples are generally fixed, dehydrated and embedded before sectioning.

Section thickness is selected according to the imaging method and scientific objective.

Paraffin sections are commonly prepared at a thickness of a few micrometres, while resin-embedded semithin sections are usually thinner and provide improved structural resolution.

Advantages

Microtome sectioning provides:

• Reproducible thin sections

• Good preservation of tissue architecture

• Compatibility with a wide range of staining and labelling methods

• Preparation of serial sections

• Reliable comparison between samples and experimental groups

• Suitable sections for qualitative and quantitative image analysis

Factors Affecting Section Quality

The quality of the sections depends on:

• Proper fixation and embedding

• Sample orientation

• Block hardness

• Blade condition and cutting angle

• Cutting speed

• Section thickness

• Tissue consistency and heterogeneity

Paraffin wax embedding is a widely used histological technique for preparing thin sections of biological tissues for light microscopy.

The method provides good preservation of tissue architecture and allows serial sections to be obtained for morphological, histochemical and immunohistochemical studies.

Before embedding, samples are usually fixed, dehydrated through graded alcohols and cleared using a solvent compatible with paraffin wax.

The tissue is then infiltrated with molten paraffin, correctly oriented in an embedding mould and allowed to solidify into a stable block suitable for microtome sectioning.

Applications

Paraffin embedding is suitable for (among others):

• Histological examination of animal and plant tissues

• Analysis of tissue organization and cellular morphology

• Routine staining procedures

• Histochemistry

• Immunohistochemistry

• In situ hybridization

• Detection of structural alterations caused by treatments or disease

• Preparation of serial sections

• Morphometric and quantitative image analysis

• Identification of regions of interest for complementary microscopy techniques

Sample Preparation

The general preparation procedure includes:

• Tissue collection and trimming

• Chemical fixation

• Dehydration through graded alcohol solutions

• Clearing with an appropriate organic solvent

• Infiltration with molten paraffin wax

• Sample orientation in an embedding mould

• Cooling and solidification of the paraffin block

• Microtome sectioning

• Collection of sections on glass slides

The exact protocol is adapted to the type, size and consistency of the sample, as well as to the intended staining or labelling procedure.

Sectioning

Paraffin blocks are sectioned using a rotary microtome equipped with a disposable or reusable blade.

Sections are generally prepared at a thickness of a few micrometres and transferred to glass slides, often using a warm water bath to flatten the sections before collection.

After drying, the sections are deparaffinized and rehydrated before staining, immunolabelling or other analytical procedures.

Main Advantages

Paraffin embedding provides:

• Good preservation of overall tissue architecture

• Production of thin and reproducible sections

• Preparation of serial sections

• Easy storage of paraffin blocks for long periods

• Compatibility with numerous histological stains

• Compatibility with many immunohistochemical techniques

• Suitable sections for qualitative and quantitative image analysis

• Relatively rapid and cost-effective processing

Limitations

Paraffin embedding involves exposure to heat and organic solvents, which may extract lipids, alter soluble components or reduce the accessibility of some antigens.

Other limitations include:

• Lower preservation of fine ultrastructure compared with resin embedding

• Possible tissue shrinkage or distortion

• Loss of lipid-rich components during processing

• Requirement for antigen-retrieval procedures in some immunohistochemical studies

• Difficulty processing very hard, highly fibrous or calcified samples without additional treatment

• Incompatibility with live-cell imaging

The balance between morphological preservation and antigen retention must be considered when selecting the fixation and processing protocol.

Factors Affecting Embedding Quality

The quality of paraffin embedding may be influenced by:

• Type and duration of fixation

• Sample size and thickness

• Dehydration conditions

• Clearing efficiency

• Paraffin infiltration time

• Processing temperature

• Sample orientation

• Paraffin hardness

• Blade condition

• Section thickness

Incomplete dehydration, clearing or paraffin infiltration may result in poorly supported tissues, tearing, folds or uneven sectioning.

Brightfield microscopy is one of the most widely used optical microscopy techniques for the observation of biological and material samples. It produces an image in which the specimen appears darker or more contrasted against a bright background.

The technique is particularly suitable for stained samples, naturally pigmented specimens and samples with sufficient intrinsic contrast.

Principle

In brightfield microscopy, transmitted white light passes through the specimen and is collected by the microscope objective. Differences in light absorption, scattering and refractive properties within the sample generate image contrast.

Transparent biological specimens often require staining because many cellular structures absorb very little visible light and may otherwise be difficult to distinguish.

Main Applications

Brightfield microscopy is commonly used for:

• General observation of cells, tissues and microorganisms.

• Histological analysis of tissue sections.

• Examination of paraffin- and resin-embedded samples.

• Observation of stained plant and animal tissues.

• Morphological analysis of cells and cellular structures.

• Evaluation of tissue organisation and pathology.

• Observation of bacteria, fungi, yeasts and parasites.

• Cell counting and assessment of cell density.

• Analysis of plant anatomy, including roots, leaves, stems and seeds.

• Examination of microtome and ultramicrotome semi-thin sections.

• Quality control of sample preparation before electron microscopy.

• Documentation of cytochemical, histochemical and immunohistochemical staining.

Common Staining Methods

Brightfield microscopy can be combined with a wide variety of staining techniques, including:

• Haematoxylin and eosin staining.

• Toluidine blue staining.

• Safranin and Fast Green staining.

• Periodic acid–Schiff staining.

• Gram staining.

• Giemsa staining.

• Histochemical and cytochemical reactions.

• Enzyme-based immunohistochemistry.

• Chromogenic in situ hybridisation.

• Metal and mineral localisation stains.

The choice of staining method depends on the sample type and the structures or molecules of interest.

Advantages

• Simple and widely accessible imaging method.

• Suitable for routine examination of stained samples.

• Rapid image acquisition.

• Low phototoxicity compared with fluorescence microscopy.

• Compatible with permanent preparations.

• Useful for large tissue areas and general morphological assessment.

• Allows colour documentation of histological and cytochemical stains.

• Compatible with digital image acquisition and quantitative image analysis.

• Useful for screening samples before more advanced microscopy techniques.

Limitations

• Low contrast in unstained transparent specimens.

• Limited ability to distinguish structures with similar optical properties.

• Lower axial resolution than confocal microscopy.

• Limited optical sectioning capability.

• Not suitable for highly specific molecular localisation unless combined with chromogenic labelling.

• Thick samples may produce overlapping structures and reduced image clarity.

• Staining procedures may introduce artefacts or alter tissue morphology.

Sample Types

Brightfield microscopy can be applied to:

• Paraffin sections.

• Resin semi-thin sections.

• Frozen sections.

• Plant tissues.

• Animal tissues.

• Cell cultures.

• Microorganisms.

• Smears and cytological preparations.

• Histological and pathological samples.

• Stained whole-mount specimens.

• Material and particle samples with sufficient optical contrast.

Sample Preparation Considerations

Sample preparation depends on the type of specimen and the intended analysis. Important considerations include:

• Selecting an appropriate fixation method.

• Preserving tissue morphology.

• Choosing a suitable embedding medium.

• Obtaining sections of appropriate thickness.

• Using staining procedures compatible with the target structure.

• Avoiding overstaining or uneven staining.

• Including appropriate positive and negative controls.

• Using mounting media that preserve colour and image quality.

• Minimising folds, tears, compression and other sectioning artefacts.

Brightfield microscopy provides a reliable and versatile method for routine morphological analysis, histology and sample quality assessment. It is often used as a first imaging step before applying more advanced techniques such as fluorescence, confocal, Raman or electron microscopy.