- GFP cages may lead to new robust fluorophores
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
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
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.
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