For well over 100 years, all microscopes were constrained by a fundamental limitation: that a given type of radiation cannot be used to probe structural details much smaller than its own wavelength. A limit to the resolution of a light microscope was therefore set by the wavelength of visible light, which ranges from about 0.4 μm (for violet) to 0.7 μm (for deep red). In practical terms, bacteria and mitochondria, which are about 500 nm (0.5 μm) wide, are generally the smallest objects whose shape we can clearly discern in the light microscope; details smaller than this are obscured by effects resulting from the wavelike nature of light. To understand why this occurs, we must follow the behavior of a beam of light as it passes through the lenses of a microscope (Figure 9–3).
Because of its wave nature, light does not follow the idealized straight ray paths that geometrical optics predicts. Instead, light waves travel through an optical system by many slightly different routes, like ripples in water, so that they
Figure 9–1 A sense of scale between living cells and atoms. each diagram shows an image magnified by a factor of ten in an imaginary progression from a thumb, through skin cells, to a ribosome, to a cluster of atoms forming part of one of the many protein molecules in our body. atomic details of biological macromolecules, as shown in the last two panels, are usually beyond the power of the electron microscope. While color has been used here in all the panels, it is not a feature of objects much smaller than the wavelength of light, so the last five panels should really be in black and white.
limit of limit of super-conventional 0.1 nm(1 A)
Figure 9–2 Resolving power. sizes of cells and their components are drawn on a logarithmic scale, indicating the range of objects that can be readily resolved by the naked eye and in the light and electron microscopes. note that new superresolution microscopy techniques, discussed in detail later, allow an improvement in resolution by an order of magnitude compared with conventional light microscopy.
interfere with one another and cause optical diffraction effects. If two trains of waves reaching the same point by different paths are precisely in phase, with crest matching crest and trough matching trough, they will reinforce each other so as to increase brightness. In contrast, if the trains of waves are out of phase, they will interfere with each other in such a way as to cancel each other partly or entirely (Figure 9–4). The interaction of light with an object changes the phase relationships of the light waves in a way that produces complex interference effects. At high magnification, for example, the shadow of an edge that is evenly illuminated with light of uniform wavelength appears as a set of parallel lines (Figure 9–5), whereas that of a circular spot appears as a set of concentric rings. For the same reason, a single point seen through a microscope appears as a blurred disc, and two point objects close together give overlapping images and may merge into one.
the following units of length are commonly employed in microscopy: μm (micrometer) = 10–6 m nm (nanometer) = 10–9 m Å (Ångström unit) = 10–10 m
Figure 9–3 A light microscope.
(a) Diagram showing the light path in a compound microscope. Light is focused on the specimen by lenses in the condenser. a combination of objective lenses, tube lenses, and eyepiece lenses is arranged to focus an image of the illuminated specimen in the eye. (B) a modern research light microscope. (B, courtesy of Carl Zeiss Microscopy, gmbh.)
Although no amount of refinement of the lenses can overcome the diffraction limit imposed by the wavelike nature of light, other ways of cleverly bypassing this limit have emerged, creating socalled superresolution imaging techniques that can even detect the position of single molecules.