Fluorescence microscopy is currently probably the most widely used microscopy technique as it enables the molecular composition of the structures being observed to be identified through the use of fluorescently-labelled probes of high chemical specificity such as antibodies. The technique of immunofluorescence is now established as one of the standard weapons in the biologist's armory. However, its use is mainly confined to studies of fixed specimens because of the difficulties of introducing antibody complexes into living specimens. For proteins that can be extracted and purified in reasonable abundance, these difficulties can be circumvented by directly conjugating a fluorophore to a protein and introducing this back into a cell. It is assumed that the fluorescent analogue behaves like the native protein and can therefore serve to reveal the distribution and behavior of this protein in the cell.
An exciting new development in the use of fluorescent probes for biological studies has been the development of the use of naturally fluorescent proteins as fluorescent probes. The jellyfish Aequorea victoria produces a naturally fluorescent protein known as green fluorescent protein (GFP). The gene for this protein has been cloned and can be transfected into other organisms. This can provide a very powerful tool for localizing regions in which a particular gene is expressed in an organism, or in identifying the location of a particular protein. Surprisingly, in many cases these chimeric proteins preserve their original function. It is therefore often possible to use this technique to visualize the intracellular distribution of a cytoskeletal protein. The beauty of the GFP technique is that living, unstained samples can be observed. There are presently several variants of GFP which provide spectrally separable emission colors (Heim and Tsien, 1996. Curr Biol 6(2):178).
Fluorescent probes that monitor the physiological state of a cell
In addition to specific labels, probes have been developed which respond to the physiological state of a cell, such as the internal ion concentration or membrane potential. Fluorescent molecules have been developed which change their optical properties in response to changes in specific aspects of their environment. For example, the dye Indo-1 exhibits a spectral shift in its fluorescent emission when bound to calcium ions. Such molecules are known as indicator molecules as they may be used to monitor the concentration of the molecule to which they are sensitized. Fluorescent indicators currently exist for calcium, pH, ATP, membrane potential, and several neurotransmitters . When cells are preloaded with an indicator for a physiologically significant molecule, fluorescence microscopy may be used to measure the intracellular distribution of that molecule. This is a very powerful technique as it can allow dynamic signaling events to be visualized in living tissue. An exciting recent development has been the development of a free-calcium reporter based on GFP. Two color variants of GFP have been engineered into a single chimeric molecule that contains calmodulin, a calcium binding protein (Miyawaki et al., 1997 Nature 388(6645):882). The chimeric molecule has been so engineered that, upon binding calcium the fluorescent peptides undergo fluorescence resonance energy transfer (FRET); the emission from the shorter wavelength fluorescent peptide is quenched and energy is transferred to excite the longer wavelength peptide thus changing the ratio of the two emitted wavelengths. The result is a a probe-concentration insensitive calcium indicator. The beauty of this technique is that the indicator does not have to be loaded into the cell under study (often a problem). Instead, the organism is transfected with the gene so all cells within the organism (for which the gene promoter is appropriate) will express the calcium indicator.
Problems with Fluorescence Microscopy
Simple fluorescence microscopy only works well with very thin specimens or when a thick specimen is cut into sections. The reason for this is that structures above and below the plane of focus give rise to interference in the form of out-of-focus flare. However, this can be removed by optical sectioning techniques. The other major problem with fluorescence microscopy is phototoxicity (Hockberger et al. 1999 PNAS, in press; also see Cornell site). When a fluorophore (endogenous or exogenous) is excited, there is a probability that, instead of decaying to a singlet state and emitting a fluorescence photon, intersystem crossing will occur to a triplet state. These long-lived states are very reactive and can damage living cells and bleach the fluorophore. One of the most significant damage mechanisms is the generation of highly reactive singlet oxygen from triplet state. When a specimen is being observed in a fluorescence microscope, the fluorophore is excited throughout the bulk of the sample, even though only one focal plane is being observed at any time. Most of the phototoxic load in a live specimen therefore comes from regions away from the thin focal plane being observed.