Collaborative work among research groups at the University of Genoa, the University of Parma, and the University of Milano-Bicocca, all in Italy, has demonstrated that polyelectrolyte cages can modify the fluorescence properties of GFP. The effort is part of an overall investigation to develop a fluorophore that has robust fluorescence approaching that of a quantum dot and that provides other benefits for biological applications. The researchers conducted three sets of experiments using a triple GFP mutant called GFPmut2 that exhibits enhanced fluorescence emission and increased protein yield when compared with wild-type GFP. The group tested the mutant in particles made of two polyelectrolytes: poly(styrenesulfonate sodium salt) (PSS) and poly(allylamine hydrochloride) (PAH). Researchers have determined that caging GFP using poly(styrenesulfonate sodium salt) (PSS) [red] and poly(allylamine hydrochloride) (PAH) [blue] influences the fluorescence properties of the protein. They created a GFP polyelectrolyte particle that is cylindrical and formed around a core with GFP in either an internal or external configuration. They also tested it with PSS and PAH individually spin-coated onto glass to create a GFP-polyelectrolyte filament, and they tested it inside the core of amorphous calcium carbonate and entrapped in wet silica gel. Reprinted with permission of Optics Express. The GFP-polyelectrolyte particle is cylindrical and has GFP that is either turned toward the inside of the particle (internal configuration) or turned outward (external configuration). The investigators also tested it with PSS and PAH individually spin-coated onto glass to create a GFP-polyelectrolyte filament inside a core of amorphous calcium carbonate and entrapped it in wet silica gel. “Biocompatible matrices allow us to study the photophysical and functional behavior [of GFP] in adjustable conditions that can be used to create a better understanding of the way [GFP] functions in different environments,” explained Alberto Diaspro of the University of Genoa. The experiments demonstrate that the caged configurations increased the brightness as well as the time it took to photobleach when compared with GFP in wet silica or inside the core of the amorphous calcium carbonate. For imaging, the researchers used a single-molecule fluorescence setup for two-photon excitation with a mode-locked Ti:sapphire laser from Spectra-Physics. For detection, they used two avalanche photodiodes from EG&E (now PerkinElmer Inc.). Diaspro said that the group chose two-photon excitation because, among other advantages, certain types of GFP have a very sharp response in terms of switchability and spatial localization under this type of excitation. Two-photon excitation also makes long-term imaging possible because of lower levels of photodamage than other methods. “There is the possibility of more efficient control of the photophysical properties [using] two-photon excitation,” he said. A version of the external configuration that had the GFP tucked inside folds of PSS had the highest brightness, an average fluorescence lifetime decay of 3.4 ns, and a time to photobleaching of 250 s. A similar version of the internal configuration had about the same brightness but somewhat longer photobleaching time (255 s). Interestingly, when the GFP was spin-coated to PSS alone to form a fluorescent filament, the fluorescence parameters were equivalent to both the GFP particles. Diaspro said that the scientists plan to work to tighten the GFP cage, which may improve the fluorescence. He added that they plan to develop nanotracers for tracking cellular events and to explore employing GFP cages to develop three-dimensional optical memory.