INTRODUCTION
Biochemical and molecular analyses are frequently complemented by microscopic examination of tissue, cell or organelle preparations. Examples range from relatively simple observations by eye to more complex experimental manipulations and measurements. For example, the evaluation of the integrity of samples during an experiment; mapping the fine details of the spatial distribution of macromolecules within cells; direct measurements of biochemical events within living tissues or the elucidation of the macromolecular structure of organelles. In the recent past, the measurement of cellular dynamics has entered a new realm of sophistication and accuracy with the introduction of super-resolution techniques that have enabled the direct viewing of macromolecular dynamics in living cells (Section 11.4.7).
There are two fundamentally different types of microscope: the light microscope and the electron microscope (Figure 11.1). Light microscopes use a series of glass lenses to focus light as it interacts with a specimen in order to form an image, whereas electron microscopes use electromagnetic lenses to focus a beam of electrons. Light microscopes are able to magnify to a maximum of approximately 1500 times, whereas electron microscopes are capable of magnifying to a maximum of approximately 200 000 times.
Magnification is not the best measure of a microscope, however. Rather, resolution, the ability to distinguish between two closely spaced points in a specimen, is a much more reliable estimate of a microscope's utility. Standard light microscopes have a lateral resolution limit of about 0.5 micrometres (μm) for routine analysis. In contrast, electron microscopes have a lateral resolution of up to 1 nanometre (nm). Computer enhancement methods have improved upon the theoretical maximum 0.5 μm resolution limit of the light microscope down to 0.1 μm resolution in some specialised instruments called s uper-resolution microscopes (Section 11.4.7).
Applications of the microscope in biomedical research can be relatively simple and routine; for example, a quick check of the status of a preparation during an experiment or of the health of cells growing on a plastic dish in tissue culture. Here, a simple bench-top light microscope is perfectly adequate. Other applications may be more involved, for example, measuring the concentration of calcium in a living embryo over a millisecond timescale. In this case, a more advanced light microscope (often called an imaging system) is required.