Figure 9–12 Fluorescence and the fluorescence microscope. (a) an orbital electron of a fluorochrome molecule can be raised to an excited state following the absorption of a photon. Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength. too much exposure to light, or too bright a light, can also destroy the fluorochrome molecule, in a process called photobleaching. (B) in the fluorescence microscope, a filter set consists of two barrier filters (1 and 3) and a dichroic (beam-splitting) mirror (2). this example shows the filter set for detection of the fluorescent molecule fluorescein. high-numerical-aperture objective lenses are especially important in this type of microscopy because, for a given magnification, the brightness of the fluorescent image is proportional to the fourth power of the numerical aperture Figure 9–14 Different fluorescent probes can be visualized in the same cell. in this composite micrograph of a cell in mitosis, three different fluorescent probes have been used to label three different cellular components (Movie 9.1). the spindle microtubules are revealed with a green fluorescent antibody, centromeres with a red fluorescent antibody, and the Dna of the condensed chromosomes with the blue fluorescent dye Dapi. (Courtesy of kevin F. sullivan.) (see Figure 9–13) but, like many organic fluorochromes, they fade fairly rapidly when continuously illuminated. More stable fluorochromes have been developed based on inorganic chemistry. Tiny crystals of semiconductor material, called nanoparticles, or quantum dots, can be excited to fluoresce by a broad spectrum of blue light. Their emitted light has a color that depends on the exact size of the nanocrystal, between 2 and 10 nm in diameter, and additionally the fluorescence fades only slowly with time (Figure 9–15). These nanoparticles, when coupled to other probes such as antibodies, are therefore ideal for tracking molecules over time. If introduced into a living cell, in an embryo for example, the progeny of that cell can be followed many days later by their fluorescence, allowing cell lineages to be tracked.
Figure 9–15 Fluorescent nanoparticles or quantum dots. (a) Quantum dots are tiny particles of cadmium selenide, a semiconductor, with a coating to make them water-soluble. they can be coupled to protein molecules such as antibodies or streptavidin and, when introduced into a cell, will bind to a target protein of interest. Different-sized quantum dots emit light of different colors—the larger the dot, the longer the wavelength—but they are all excited by the same blue light. Quantum dots can keep shining for weeks, unlike most fluorescent organic dyes. (B) in this cell, microtubules are labeled (green) with an organic fluorescent dye (alexa 488), while a nuclear protein is stained (red) with quantum dots bound to streptavidin. on continuous exposure to strong blue light, the fluorescent dye fades quickly while the quantum dots continue to shine. (C) in this cell, the labeling pattern is reversed; a nuclear protein is labeled (green) with an organic fluorescent dye (alexa 488), while microtubules are labeled (red) with quantum dots. again, the quantum dots far outlast the fluorescent dye. (B and C, from L. Medintz et al., Nat. Mater. 4:435–446, 2005. With permission from Macmillan publishers Ltd.)
Later in the chapter, additional fluorescence microscopy methods will be discussed that can be used to monitor changes in the concentration and location of specific molecules inside living cells.
antibodies Can Be Used to Detect specific Molecules