Micrograph of human cells modified to act as metal sensors. Researchers at JILA built hardware to help time the dynamic states of these sensors, which change fluorescence color when they bind metal ions. The cells are false-colored to indicate the extent of their reactions; the darker cells have bound the most metal ions. Image: Hairong Ma/JILA
Individual cells modified to act as sensors using
fluorescence are already useful tools in biochemistry, but now they can add
good timing to their resumé, thanks in part to NIST.
With the added capability to track the timing of dynamic
biochemical reactions, cell sensors become more useful for many studies, such
as measurements of protein folding or neural activity.
As described in the Journal of the American Chemical
Society, a NIST biophysicist working at JILA and a collaborator at the
University of Colorado Boulder (CU) developed a microfluidic system that
records biochemical reactions over a time span of milliseconds to seconds in
living human cells modified to act as FRET (fluorescence resonance energy
The fast, flexible system uses lasers to measure sensor
signals at two points in time at a rate of up to 15 cells per second.
Statistical data, such as the average value of the FRET response for thousands
of cells, can be collected in minutes.
“Our system is the first one that measures FRET
response times at the single-cell level, while at the same time measuring over
many cells,” says JILA Fellow Ralph Jimenez, whose research group built
the optics, microfluidics, electronics and other hardware.
JILA is a joint institute
of NIST and CU. Jimenez
is collaborating with Amy Palmer, an assistant professor in CU’s Department of
Chemistry and Biochemistry, who handled the molecular design and cell-biology
aspects of the project.
The FRET technique relies on reactions that occur between
large biological molecules in close proximity to each other. One molecule
absorbs light energy from a laser and transfers this energy to the nearby
acceptor molecule. The acceptor molecule then releases this energy as light
(fluorescence) at a characteristic wavelength that is different from the
original laser light. Measurements of this fluorescence indicate the extent of
the energy transfer. FRET can be used to study many types of cellular
processes. In these experiments, the researchers were interested in the type
and concentration of metal ions within cells, which can affect important cell
processes. The JILA/CU experiments used cells genetically modified to take up
particular metal ions and signal changes in their concentrations by altering
the FRET signals.
The researchers made a microfluidic device with a
flow-control valve system that mixes cells and metal-containing chemicals in
just a few milliseconds. The cells then pass single file through two blue laser
beams that excite the FRET fluorescence signal at different locations in the
device. With precise flow control and flexible device design, cell travel time
between the two locations can be varied from 1 msec to 10 sec. Scientists
measure the FRET signal changes within individual cells between the two
“FRET is an important measurement technique used in
bio-imaging, so it’s great that NIST could begin to contribute to measurements
of the fidelity of FRET-based sensors,” Jimenez says. “We have a lot
more work planned for the future with this instrument.”
The project is part of the research team’s effort to develop
cell sensors with improved optical, physical and chemical properties and to
enable detection of very faint signals in living cells.