The fluorescent dye can be covalently attached directly to the specific antibody; however, the bound antibody is more commonly detected by fluorescently labeled anti-immunoglobulin, a technique known as indirect immunofluorescence. The dyes chosen for immunofluorescence are excited by light of one wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum. The most commonly used fluorochromes are fluorescein, which emits green light; Texas Red and peridinin chlorophyll protein (PerCP), which emit red light; and rhodamine and phycoerythrin (PE), which emit orange/red light (Fig. A.11). By using selective filters, only the light coming from the fluorochrome used is detected in the fluorescence microscope (Fig. A.12). Although Albert Coons first devised this technique to
Fig. A.12 Immunofluorescence microscopy. Antibodies labeled with a fluorescent dye such as fluorescein (green triangle) are used to reveal the presence of their corresponding antigens in cells or tissues. The stained cells are examined using a microscope that exposes them to blue or green light to excite the fluorescent dye. The excited dye emits light at a characteristic wavelength, which is captured by viewing the sample through a selective filter. This technique is applied widely in biology to determine the location of molecules in cells and tissues. Different antigens can be detected in tissue sections by labeling antibodies with dyes of distinctive color. Here, antibodies against the protein glutamic acid decarboxylase (GAD) coupled to a green dye are shown to stain the β cells of pancreatic islets of Langerhans. The α cells do not make this enzyme and are labeled with antibodies against the hormone glucagon coupled with an orange fluorescent dye. GAD is an important antigen in type 1 diabetes, a disease in which the insulin-secreting β cells of the islets of Langerhans are destroyed by an immune attack on self tissues (see Chapter 15). Photograph courtesy of M. Solimena and P. De Camilli.
identify the plasma cell as the source of antibody, it can be used to detect the distribution of any protein. By attaching different dyes to different antibodies, the distribution of two or more molecules can be determined in the same cell or tissue section (see Fig. A.12).
The development of the confocal fluorescent microscope, which uses computer-aided techniques to produce ultrathin optical sections of a cell or tissue, gives very high resolution (sub-micrometer) fluorescence microscopy without the need for elaborate sample preparation. The light source for excitation (a laser) is focused onto a particular plane in the specimen, and the emitted light is refocused through a ‘pinhole’ so that only light from the desired plane reaches the detector, thus removing out-of-focus emissions from above or below the plane. This gives a sharper image than conventional fluorescence microscopy, and a three-dimensional picture can be built up from successive optical sections taken along the ‘vertical’ axis. Confocal microscopy can be used on fixed cells stained with fluorescently tagged antibodies or on living cells expressing proteins tagged with naturally fluorescent proteins. The first of these fluorescent proteins to come into wide use was green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria. The list of fluorescent proteins in routine use now includes those emitting red, blue, cyan, or yellow fluorescence. By using cells transfected with genes encoding different fusion proteins, it has been possible to visualize the redistribution of T-cell receptors, co-receptors, adhesion molecules, and other signaling molecules, such as CD45, that takes place when a T cell makes contact with a target cell (see Fig. 9.37).