Physical Address

304 North Cardinal St.
Dorchester Center, MA 02124

Introduction to microscopy


Preparation of a Glass Slide

Histology is about looking at structure, and in this introductory section we aim to provide some guidelines to assist the absolute beginner in examining and interpreting the images in this book. Examination of any biological tissue under a microscope requires a number of preparation steps. These complex techniques are performed by highly skilled professionals known as biomedical scientists who are key members of diagnostic pathology and medical research laboratories. Details of these techniques are outwith the scope of this text, however a brief overview is given below.

Further information is also provided in Chapter 1 of Wheater’s Pathology (6th edition). Once any fresh tissue (human or animal) is removed, it will generally require the following steps to produce a glass slide for examination under a light microscope. This is vital to ensure the tissue is preserved and remains firm enough for processing, cutting and staining, and also to preserve the slide so that it can be stored and viewed as and when required. Some of these steps are illustrated below and are described in greater detail at the end of this appendix.

  • 1)

    FIXATION and TRIMMING

    • Fixation of fresh tissues is essential to prevent autolysis (breaking down) of the tissues. Formalin solutions are normally used for this purpose and help to preserve the tissues for processing and cutting (see below).

    • Once fixed, the area of interest in the tissue can be trimmed (dissected) so that it will fit onto a glass slide. Smaller samples do not require this step.

  • 2)

    PROCESSING and EMBEDDING

    • Processing includes the steps involved between fixing the tissues and impregnating the tissues with paraffin wax. These steps can be carried out by hand but are most commonly performed using a processor machine. The tissue is dehydrated in graded alcohol solutions.

    • Embedding is when the processed tissue is infiltrated by hot liquid paraffin wax and placed in a mould. When cool, this solidifies into a block which provides a firm cutting surface for the next step.

  • 3)

    MICROTOMY

    • Very thin slices of tissue (3–5 μm) can be cut from this solid wax block using a special knife called a microtome. These thin tissue sections are floated on a water bath and then mounted on a glass slide.

  • 4)

    STAINING AND COVER SLIPPING

    • The tissue section is colourless at this point as illustrated and requires staining with special tissue dyes or antibodies in order to view the tissues under a microscope. These stains are further discussed in Appendix 2 .

    • A protective glass cover (cover slip) is applied to protect the underlying tissue.

  • 5)

    FINAL PRODUCT

    • This is a skin sample which has been stained with haematoxylin and eosin, commonly referred to as ‘H&E’. Three tissue sections have been provided on the same slide, allowing easy examination under the microscope.

Microscopy

Light microscopy

Light microscopes are an essential tool for most histologists, scientists and pathologists around the world. For histology in particular, light microscopy (LM) has revolutionised our ability to study the structure and function of cells and tissues at high resolution. Many of the images illustrated in this book have been derived from light microscopes. The function of LM is based on the principles of the refractile quality of light when passed through a lens or series of lenses. There are many different types of light microscope, including brightfield, phase contrast and confocal, which allow examination of fixed tissues (see above), organisms or even live cells such as those in cell culture.

In basic terms, light microscopes function by using a beam of light which, when focused through a specimen, produces an image that is then magnified using a lens or objective. With a compound microscope, there are two or more lenses which can further magnify the image. Finally, the image can be viewed by eye using an eyepiece. The component parts of a compound light microscope and the basic steps in setting up a microscope are illustrated below.

  • SETTING UP A LIGHT MICROSCOPE

    • A)

      SET EYEPIECES

      • 1)

        Turn on the illumination switch, taking care not to set the light too high.

      • 2)

        Ensure objective lens (×4 or ×10) is in place.

      • 3)

        Adjust the interpupillary distance between the eyepieces by hand so that the light circles seen through the eyepieces with each eye merge to form one circle.

      • 4)

        Place a slide on the stage, coverslip upwards

      • 5)

        Focus using the coarse (large) and then the fine (smaller) focus by closing one eye so that the image appears sharp.

      • 6)

        Accommodate the other eye by turning the diopter ring on the eyepiece.

      • 7)

        Repeat the focussing process using the high power ×40 lens.

    • B)

      SET CONDENSER

      • 1)

        Close the diaphragm aperture so that the light field is small.

      • 2)

        Focus the field diaphragm using the condenser focus.

      • 3)

        Centre the field diaphragm using the centre adjustment dials on the condenser (see image below).

  • 4)Repeat for each objective to check the field diaphragm is centred in each.

  • C) SET CONTRAST

    • 1)

      Remove one eyepiece and look down the tubing.

    • 2)

      Turn the aperture switch up full then reduce by approximately 25% so that aperture diaphragm is just inside the field of the objective.

  • Key:

  • A – ocular lens (eyepiece)

  • B – diopter adjustment

  • C – objective lenses

  • D – stage

  • E – centre adjustment dials

  • F – condenser iris adjustment

  • G – field diaphragm adjustment

  • H – light switch

  • I – fine focus adjustment

  • J – coarse focus adjustment

  • K – condenser

Digital microscopy

It is now possible to digitally scan and view histology slides on a high resolution computer screen rather than using a traditional light microscope. These platforms are particularly useful for educational purposes, especially where online learning is required. Many pathology laboratories now use digital pathology instead of microscopes as the primary method for reviewing slides. The technology also facilitates image analysis and is especially valuable in research settings.

Light microscopy versus electron microscopy

This book mainly uses photomicrographs taken with the light microscope ( LM ) (colour images) and the electron microscope ( EM ) (black and white images). Simply put, the LM and EM differ in optical resolution and available magnification . In practical terms, ‘resolution’ refers to the capacity of an optical system to reveal detail in a specimen. The resolution available from a conventional LM is only about 0.2 μm. Thus, at distances of less than 0.2 μm, objects that are actually separate from one another will appear to merge. In contrast, EM resolution for biological specimens is as little as 1 nm, so that the resolving power is about 200-fold better than LM. In addition, maximum ‘available magnification’ is limited to about ×1000 in most student LMs, whereas an EM readily achieves 100-fold greater magnification, or about ×100 000. EM images are therefore said to display cell and tissue ultrastructure .

EM images may be two-dimensional or three dimensional

There are two types of electron microscope: scanning EM and transmission EM . Scanning EM produces three-dimensional (3D) images, but these are restricted to the surface of the object, with the internal structure concealed from view. Transmission EM is so named because the electron beam must pass through the specimen to form an image. To achieve this, ultrathin sections (50–100 nm) must be cut. Transmission of the electron beam through the tissue results in a two-dimensional (2D) image of the plane of the section. In practice, transmission EM is more informative of biological ultrastructure, and these images predominate in this book. We have supplemented these with scanning EM images where it helps with 3D conceptualisation (see Fig. 16.13 ). As a matter of convention, the abbreviation EM can be assumed to be a transmission EM, while we have identified scanning EMs as SEM.

Light and electron microscopy are complementary

The strengths of LM and EM differ yet complement one another very effectively. With LM, one can observe large areas of a specimen (usually several cm 2 ). A wide range of staining methods, some empirical, some specific, are available for LM, permitting identification of cell and tissue features; many of these stains are polychromatic, i.e. they produce multiple colours in the specimen which, besides looking pretty, help to identify different components. For certain specimens, sections slightly thicker than usual may be used to demonstrate 3D features. Thus from LM, students can expect to gain an understanding of overall cell and tissue architecture.

The superior resolution and magnification of EM permit visualisation of many features which simply cannot be seen by LM. Yet in some respects, EM is less flexible than LM. For example, the available area in EM specimens is generally less than 1 mm 2 and this may make it difficult to obtain representative fields. Few staining methods are available for EM and these produce only monochromatic (black and white) images. EM is also costly and time-consuming and usually not available to the average student.

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here

Related Posts