Introduction to microscopy
Introduction to microscopy

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Introduction to microscopy

5 Interpreting the appearance of sections

Most histological sections are two-dimensional slices, from a three-dimensional piece of tissue. Exactly what will be seen on the microscope slide depends on the plane of the section, i.e. the position of the microtome cut, in relation to the anatomical structures in the tissue. Most tissues are cut in a plane, which allows the underlying structure of that tissue to be seen and interpreted most easily. For example, sections of the intestine and skin are usually cut in transverse section, so that all layers of the tissue are visible. Positioning the tissue before embedding and knowing its orientation are therefore critically important, so that the sectioning is done in the correct plane. For biopsy specimens, there is no option for deciding the plane of the section, as the direction of the needle tract determines what tissue is available and how it is orientated. Pathologists expect to view sections in their best orientation as this then gives them the best opportunity of identifying abnormalities.

Many anatomical structures are not readily visible in thin, two-dimensional sections, simply because most of the structure is not present. In this case the three-dimensional structure needs to be interpreted from what can be seen. In order to give you some insight into this problem, take the three structures shown in Figure 10 and make simple sketches of how they would appear if sectioned in the planes indicated.

Figure 10 Understanding the plane of the section: make simple diagrams of the structures that would be seen if the following tissue or cell were sectioned in the planes indicated (A-C). Note that on the neuron, the thickness of the section is significant in relation to the thickness of the cell.

This exercise should make you appreciate that it is not just the orientation of the section but also the position (depth) into the tissue or tissue block that affects the appearance of the structures. For example, the blood vessel can appear as one or two structures, depending on the level of the section within the block. For this reason, it may be necessary to section a block at different levels to obtain suitable slides. (The initial cut-up of the tissue should have ensured that the lesion is within the block so it should not be necessary to hunt through the block to find it.)

Another problem in interpreting sections is that some structures (e.g. the neuron in Figure 10) are too big to be seen in a thin section - much of the cell and its overall architecture cannot be seen. One solution is to cut thicker sections.

SAQ 7

List three considerations that might apply to the preparation and interpretation of thicker sections, which do not apply to regular 3µm sections.

Answer

The staining procedure might require longer to allow the stain to permeate the tissue; this problem becomes more acute with immunohistochemistry, since antibodies are large proteins which take much longer to diffuse into thick sections than thin ones.

Destaining, stages may also take longer, especially since more dye will be absorbed by the thicker section, making it darker and the structural detail more difficult to distinguish.

Thin sections typically are only one or two cells deep, so it is possible to distinguish cellular detail well. In thick sections, some cells will overlie others so that distinguishing details of individual cells or the tissue structure may be more difficult.

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