TECHNIQUES USED IN HISTOLOGY AND CELL BIOLOGY

Neurobiology 104 - 2003-09-23 Dr Geoff Meyer

Examining thin sections of tissue using the light microscope will be your major tool to study cell/tissue and organ morphology. An account of how thin sections of tissue are obtained is provided in your lab manual and computer program - but in summary:

(a) Tissue is removed and immersed in a preservative solution (fixative).

(b) Since tissues are composed of a large amount of water, that water is removed by dehydration.

(c) Tissue is embedded in a suitable medium.

(d) Thin sections of tissue (5-10µ) are cut using a microtome.

(e) Sections are mounted on glass slides.

(f) Sections are then stained.

(g) A coverslip is permanently affixed using a suitable mounting medium.

(h) Your section is ready to examine with the light microscope (with limitations!).

Stains

Hematoxylin and Eosin (H&E) are the most commonly used stains. They differentiate between acidic and basic components of the cell. Hematoxylin is a base and colours acidic components (eg. DNA and RNA in the nucleus) of a cell blue/purple. RNA is then referred to a “basophilic”. Eosin is an acid and colours basic components (eg. cytoplasmic components having a “basic pH” of a cell) a pinkish colour. These basic components are then referred to as “acidophilic”.

With some stains, some tissue/components are said to be “metachromatic” and the stain (eg. toluidine blue) is said to exhibit “metachromasia”.

Some stains differentiate fibrous components of the extracellular matrix and metallic salts that precipitate on tissues form metallic deposits on them.

Light microscopy

Utilises specific arrangements of lenses to magnify an image.

An electric bulb is the light source used to illuminate the tissue section. The image (gathered by any one of 4 objective lenses) is further magnified by the ocular lens of the eyepiece. This lens focuses the image on the retina of the eye. The total magnification of the image depends on the magnifying powers of the objective lens and that of the ocular lens – usually 40X-1000X.

Resolution

Quality of the image depends not only on magnifying capability of the lens but also on its resolution – its ability to show that 2 distinct objects are separated (by a distance).

*There are different types of light microscopes.

Electron microscopy

Using an electron beam instead of light allows resolution of structures as small as 1nm (ie. subcellular morphology) in well prepared tissues.

1. Better fixation methods.

2. Small tissue fragments.

3. Embedded in a robust material.

4. Extremely thin sectioning.

5. Stained with heavy metals.

Scanning microscopy

Provides 3D views of the surface of cells/tissues. The tissue is coated in gold and an electron beam scans the surface. Electrons scattered/ejected from the surface are collected and used to reconstruct a fine 3D representation of the surface. If cells are frozen, treated with cryopreservatives then fractured there is a tendency for the fractures to occur along cleavages planes in regions of least molecular bonding (ie. between inner and outer leaflets of membranes) and so the macromolecular structure of the internal aspects of membranes is revealed.

Histochemistry

This method provides information about the presence and location of intracellular and extracellular molecules. Histochemistry is performed mostly on frozen tissue and relies on the chemical reactivity, enzymatic activity or other physiochemical activity associated with the cell component of interest. Reactions of interest are combined with dyes and take on certain discerning colours.

Immunocytochemistry

More precise intracellular and extracellular localisation of macromolecules can be achieved using immunocytochemistry. An antibody against a particular macromolecule is created and localised by reaction with the macromolecule then visualised by labelling the antibody with a fluorescent dye or horseradish peroxidase.

Autoradiography

Radioactive metabolites are taken up by cells and detected by dipping sections in a photographic emulsion and allowing the radioactivity to create silver grains in the emulsion. An example is the incorporation of radiolabelled thymidine (a DNA component) into tissues. The autoradiograph will show cells that are actively dividing. Specific temporal events can be determined by injection of radiolabelled compound into an animal and taking tissue specimens at selected time intervals.

Cell culture

Cells may be grown in artificial media and enable the structural and functional attributes of cells to be examined.

Cell fractionation

Allows cells to be disrupted and components to be isolated by high speed centrifugation and examined.

Digital Imaging techniques - Confocal Laser Scanning Microscopy

(CLSM) is a relatively new light microscopical imaging technique (introduced around 1980 by M. Petran and A. Boyde). The primary value of the CLSM to the biologist is its ability to produce optical sections through a 3-dimensional (3-D) specimen - e.g., an entire cell or a piece of tissue - that, to a good approximation, contain information from only one focal plane. Therefore, by moving the focal plane of the instrument step by step through the depth of the specimen, a series of optical sections can be recorded. This property of the CLSM is fundamental for solving 3-D biological problems where information from regions distant from the plane of focus can obscure the image (thick objects). As a valuable by-product, CLSM produces digital images which are amenable to image analysis and processing, and can also be used to compute surface- or volume-rendered 3-D reconstructions of the specimen.