FLUORESCENCE MICROSCOPY and GFP
Light microscopy has been brought to the level of molecular analysis by methods for labeling specific molecules so that they can be visualized within cells. Specific genes or RNA transcripts can be detected by hybridization with nucleic acid probes of complementary sequence, and proteins can be detected using appropriate antibodies (see Chapter 4). Both nucleic acid probes and antibodies can be labeled with a variety of tags that allow their visualization in the light microscope, making it possible to determine the location of specific molecules within individual cells.
Figure 1.29 Fluorescence microscopy (A) Light passes through an excitation filter to select light of the wavelength (e.g., blue) that excites the fluorescent dye. A dichroic mirror then deflects the excitation light down to the specimen. The fluorescent light emitted by the specimen (e.g., green) then passes through the dichroic mirror and a second filter (the barrier filter) to select light of the wavelength emitted by the dye. (B) Fluorescence micrograph of a newt lung cell in which the DNA is stained blue and microtubules in the cytoplasm are stained green.
Fluorescence microscopy is widely used to study the intracellular distribution of molecules (Figure 1.29). A fluorescent dye is used to label the molecule of interest within either fixed or living cells. The fluorescent dye is a molecule that absorbs light at one wavelength and emits light at a second wavelength. This fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye and then using appropriate filters to detect the specific wavelength of light that the dye emits. Fluorescence microscopy can be used to study a variety of molecules within cells. One frequent application is to label antibodies directed against a specific protein with fluorescent dyes, so that the intracellular distribution of the protein can be determined.
Figure 1.30 Fluorescence microscopy of a protein labeled with GFP A microtubule-associated protein fused to GFP was introduced into mouse neurons in culture and visualized by fluorescence microscopy. Nuclei are stained blue.
A revolutionary advance in fluorescence microscopy came with the use of the green fluorescent protein (GFP) of jellyfish to visualize proteins within living cells. GFP can be fused to any protein of interest using standard methods of recombinant DNA, and the GFP-tagged protein can then be expressed in cells and detected by fluorescence microscopy, without the need to fix and stain the cell as would be required for the detection of proteins with antibodies. Because of its versatility, the use of GFP has become widespread in cell biology, and has been used to study the localization of a wide variety of proteins within living cells (Figure 1.30). Several related fluorescent proteins with blue, yellow, or red emissions are also available, further expanding the utility of this technique by allowing different proteins to be visualized simultaneously.