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Fluorescence imaging has become a methodological pillar of biological investigation. One reason for this is the explosion in the number and variety of fluorescent probes available for researchers. In spite of this, researchers continue to push the boundaries of what is possible with the available probes.

This continuing need for new probes with new characteristics has prompted the US National Institutes of Health to fund centers devoted to probe development. One of these centers is the Molecular Biosensor and Imaging Center at Carnegie Mellon University where Alan Waggoner, who has been developing fluorescent probes for years, is the director.

Although much fluorescent probe development and use has been focused on fluorescent proteins, Waggoner has concentrated on developing small chemical fluorophores such as the popular Cy3 and Cy5 dyes. Small chemical dyes are ideal for some applications, but the difficulty in using them to selectively label proteins in living cells is a drawback for many applications. Rather, fluorescent proteins have proven very popular for these applications.

Most fluorescent proteins, however, have the drawback that labeling requires creation and expression of hybrid fusion proteins that are constitutively fluorescent. Waggoner and his coworkers wanted a way to label target proteins in living cells at a time and place chosen by the investigator. Their concept was to combine environmentally sensitive dyes called fluorogens that are nonfluorescent in solution with protein fusion tags that can bind the dyes and induce a strong fluorescent signal. Thus, an investigator could visualize a target protein containing a tag at a desired time by applying the fluorogen to the cell.

They picked human single-chain antibodies (scFvs) as their protein tag platform to create fluorogen activating proteins (FAPs) and then selected candidate fluorogens. “We went into our treasure chest of dyes that we have had for many years and picked some that we thought we could combine with the single-chain antibodies and get substantial fluorescent signals,” says Waggoner.

Because of their extensive experience with small molecule dyes, the first one they picked, TO1, worked. This fluorogen was known to become highly fluorescent upon binding DNA. Using selection and directed evolution of the scFvs and chemical modification of TO1 they obtained a high affinity scFv-dye combination that produced a 2,600-fold fluorescence enhancement of the green-emitting fluorogen.

One of their goals is to create a library of scFv-fluorogen pairs with different spectral and physical properties. As a first step on this road they used a red fluorogen, MG, and selected FAPs that induced fluorescence up to 18,000-fold. Additional colors are likely on the way.

Both TO1 and MG are cell-impermeable, which is ideal for selectively labeling functional cell-surface receptors (Fig. 1). To complement the cell-surface labeling they also developed a cell-permeable ester of MG that labels proteins inside the cell. According to Waggoner, “While chemical modification of TO1 reduced DNA binding and nonspecific binding to cell material, MG is more suited for intracellular labeling due to its lower DNA binding.” The low fluorescence due to nonspecific binding to cellular proteins may be an advantage over a different fluorogen system based on a tetracysteine protein tag and biarsenical dyes. In contrast to the tetracysteine tag, intracellular labeling with FAPs is now limited to the cell lumen because scFvs are only partially functional in the reducing environment of the cytoplasm, but Waggoner's group thinks this limitation will likely be overcome in the future.

Figure 1: Visualization of cell-surface receptors tagged with two different FAPs and exposed to green and red fluorogens.
figure 1

Scale bar, 10 μm. Reprinted from Nature Biotechnology.

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The flexible design of their FAP system suggests that the capabilities of this class of probes have only begun to be explored, and it will likely become a valuable tool for dissecting cellular pathways.