Dr. Jörg Schwartz, firstname.lastname@example.org
PITTSBURGH – 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
The new approach provides a single compact protein tag with signal
enhancement provided by modestly enlarging only the targeted dye molecule.