27.9 *extended topic* microscopy enhanced by the wave characteristics

Learning objectives

By the end of this section, you will be able to:

• Discuss the different types of microscopes.

Physics research underpins the advancement of developments in microscopy. As we gain knowledge of the wave nature of electromagnetic waves and methods to analyze and interpret signals, new microscopes that enable us to “see” more are being developed. It is the evolution and newer generation of microscopes that are described in this section.

The use of microscopes (microscopy) to observe small details is limited by the wave nature of light. Owing to the fact that light diffracts significantly around small objects, it becomes impossible to observe details significantly smaller than the wavelength of light. One rule of thumb has it that all details smaller than about $\lambda$ are difficult to observe. Radar, for example, can detect the size of an aircraft, but not its individual rivets, since the wavelength of most radar is several centimeters or greater. Similarly, visible light cannot detect individual atoms, since atoms are about 0.1 nm in size and visible wavelengths range from 380 to 760 nm. Ironically, special techniques used to obtain the best possible resolution with microscopes take advantage of the same wave characteristics of light that ultimately limit the detail.

The most obvious method of obtaining better detail is to utilize shorter wavelengths. Ultraviolet (UV) microscopes have been constructed with special lenses that transmit UV rays and utilize photographic or electronic techniques to record images. The shorter UV wavelengths allow somewhat greater detail to be observed, but drawbacks, such as the hazard of UV to living tissue and the need for special detection devices and lenses (which tend to be dispersive in the UV), severely limit the use of UV microscopes. Elsewhere, we will explore practical uses of very short wavelength EM waves, such as x rays, and other short-wavelength probes, such as electrons in electron microscopes, to detect small details.

Another difficulty in microscopy is the fact that many microscopic objects do not absorb much of the light passing through them. The lack of contrast makes image interpretation very difficult. Contrast is the difference in intensity between objects and the background on which they are observed. Stains (such as dyes, fluorophores, etc.) are commonly employed to enhance contrast, but these tend to be application specific. More general wave interference techniques can be used to produce contrast. [link] shows the passage of light through a sample. Since the indices of refraction differ, the number of wavelengths in the paths differs. Light emerging from the object is thus out of phase with light from the background and will interfere differently, producing enhanced contrast, especially if the light is coherent and monochromatic—as in laser light.