Fluorescence detection with genetically targeted probes has massively extended the range of questions that can be addressed by biological microscopy and cytometry. There are major shortfalls to this approach, however, particularly when it comes to monitoring individual molecules, where spatial resolution and a lack of fluorescent light are issues. But now researchers have addressed these problems with the creation of fluorogen-activating probes (FAPs). The researchers, from Carnegie Mellon University’s Molecular Biosensors and Imaging Center, say that the FAPs are brighter than traditional fluorescent probes and can be used to monitor the biological activity of individual proteins in real time. Fluorescent probes (also called fluorophores) are parts of molecules that absorb light at a certain wavelength and then re-emit some of the energy at a different – but equally specific – optical frequency, depending on the chemical environment. Fluorescent proteins are particularly important for understanding how proteins interact with each other in space and time – controlling the health of human cells, for example. In contrast to traditional fluorescent proteins, which are always glowing once expressed in a cell, FAPs emit photons only when bound to a fluorogen, an otherwise nonfluorescent dye. This means that when the protein and dye bind, they emit light that can be used to track the protein on the cell surface and within living cells. The scientists say that these fluoromodules come in a spectrum of colors and are more photostable than other fluorescent proteins. Shown at top is detection using the normal FAP-fluorogen pair; at bottom, detection using the FAP-dyedron and revealing the enhanced brightness achieved with the dyedron at identical excitation power. Courtesy of Carnegie Mellon University. This offers the opportunity to trace individual proteins. “Molecules that are close together in a living cell can’t be discriminated,” said Marcel Bruchez, one of the research team leaders. “That’s just the physics of the diffraction of light,” he added, referring to the fact that the fluorescence signals from closely positioned and traditionally labeled molecules would not be distinguishable under the microscope. But with the new method, each of the FAP-labeled proteins can be activated – one at a time. This avoids interference between various proteins and offers the opportunity to determine the exact position of individual molecules. As a result, the resolution of imaging proteins improves from 200 to less than 5 nm. “We hope that this can be extended to 1 to 2 nm,” Bruchez said. The latest step is turning up the light emitted from fluoromodules. In a paper recently published online in the Journal of the American Chemical Society, the group reports a new class of fluorogenic dyes that amplify the signal emitted by their fluoromodules. “By using concepts borrowed from chemistry, the same concepts used in things like quantum dots and light-harvesting solar cells, we were able to create a structure that acts like an antenna, intensifying the fluorescence of the entire fluoromodule,” Bruchez said. To make the fluoromodules brighter, the researchers began with one of their existing probes. They took one of their standard fluorogens, malachite green, and coupled it with another dye called Cy3 to get a complex they call a “dyedron.” The dyedron is based on a special type of treelike structure called a dendron, with one malachite green molecule acting as the trunk and several Cy3 molecules acting as the branches. The two dyes have overlapping emission and absorption spectra – Cy3 typically emits energy at a wavelength where malachite green absorbs energy, and this overlap allows the dyes to efficiently transfer energy between them. When the Cy3 dye molecules become excited by a light source, such as a laser, they immediately “donate” their excitation energy to malachite green, boosting the signal emitted by the malachite green. The bright but small dye particles will allow researchers to expand live-cell imaging research without having to increase the intensity of the laser used to visualize the proteins or to label the protein being studied with numerous copies of the fluorescent tag. Both options so far come with the downside of potentially altering the biology of the system being studied, either through unwanted laser energy or the increased molecular weight caused by the multiple tags added to the protein. The new approach provides a single compact protein tag with signal enhancement provided by modestly enlarging only the targeted dye molecule.