Basic Light Microscopy

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Light microscopy is an indispensable technique for cell and molecular biologists to study cellular structures and biological processes in both living and fixed cells. This chapter provides an overview of light microscopy, describes the important parts of the microscope and goes on to explain how to set up a standard research microscope for bright field and phase contrast microscopy. There is also a short section on confocal microscopy. More comprehensive descriptions of the different forms of light microscopy are found elsewhere [1–4]. Microscopes are instruments that produce an enlarged image of a specimen. The eyepieces and the objectives are the main components of the magnification system of the microscope, the product of the magnification of the objective lens and the ocular lens give the total magnification of the microscope. The visibility of the magnified specimen depends on contrast and resolution. Contrast is the difference in light intensity between an object and its background. Some biological samples contain coloured compounds, for example pigmented animal cells and chlorophyll-containing chloroplasts in plant cells, but most biological samples are colourless and have to be fixed and stained before observation [5]. Such stained specimens are observed using bright field microscopy. Other kinds of microscope systems are available to enhance contrast in living samples; these include phase contrast, dark field, differential interference contrast (DIC) and fluorescence microscopy (Table 1.1). The flow chart in Figure 1.1 will help in the selection of the appropriate microscopic observation method.