Fluorescence Microscopy

The human eye requires contrast to perceive details of objects. Several ingenious methods of improving the contrast of microscopy images have been designed, and each of them opened new applications of optical microscopy in biology. The simplest and very effective contrasting method is ‘‘dark field.’’ It exploits the scattering of light on small particles that differ from their environment in refractive index – the phenomenon known in physics as the Tyndall effect. A fixed and largely featureless preparation of tissue may reveal a lot of its structure if stained with a proper dye – a substance that recognizes specifically some tissue or cellular structure and absorbs light of a selected wavelength. This absorption results in a perception of color. The pioneers of histochemical staining of biological samples were Camillo Golgi (an Italian physician) and Santiago Ramon y Cajal (a Spanish pathologist). They received the 1906 Nobel Prize for Physiology or Medicine. A revolutionary step in the development of optical microscopy was the introduction of phase contrast proposed by the Dutch scientist Frits Zernike. This was such an important discovery that Zernike was awarded the 1953 Nobel Prize in Physics. The ability to observe fine subcellular details in an unstained specimen opened new avenues of research in biology. Light that passes through a specimen may change polarization characteristics. This is a phenomenon exploited in polarization microscopy, a technique that has also found numerous applications in material science. The next important step in creating contrast in optical microscopy came with an invention of differential interference contrast by Jerzy (Georges) Nomarski, a Polish scientist who had to leave Poland afterWorldWar II and worked in France. In his technique, the light incident on the sample is split into two closely spaced beams of polarized light, where the planes of polarization are perpendicular to each other. Interference of the two beams after they have passed through the specimen results in excellent contrast, which creates an illusion of three-dimensionality of the object.