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Sorting good data from bad is critical when analyzing
microscopic structures like cells and their contents, according to researchers
at Rice University. The trick is to find the right window of time through which
to look.
A new paper by the Rice laboratory of Angel Martí, an
assistant professor of chemistry and bioengineering, offers a methodology to
optimize the sensitivity of photoluminescent probes using time-resolved
spectroscopy. Martí and co-author Kewei Huang, a graduate student in his group,
found their technique gave results nearly twice as good as standard
fluorescence spectroscopy does when they probed for specific DNA sequences.
Their results were reported in Analytical Chemistry.
In spectroscopy, chemicals and materials from proteins to
nanotubes can be identified and tracked by their fluorescence—the light they
return when excited by an input of energy, usually from a laser. In the kind of
targeted spectroscopy practiced by Martí and his colleagues, a luminescent
probe called a molecular beacon is designed to attach to a target like a DNA
sequence and then light up.
Improving a probe’s ability to detect ever smaller and
harder-to-find targets is important to biologists, engineers and chemists who
commonly work on the molecular scale to analyze cell structures, track disease,
or design tiny machines.
One problem, Martí says, has been that even in an experiment
lasting a fraction of a second, a spectrometer can return too much information
and obscure the data researchers actually want. “In standard fluorescence
spectroscopy, you see noise that overlaps with the signal from your probe, the
scattering from your solution or cuvettes, plus the noise from the detector,”
he says. The saving grace, he says, is that not all those signals last the same
amount of time.
Time-resolved spectroscopy provides part of the answer,
Martí says. Compared with standard spectroscopy, it’s like taking a film
instead of a snapshot. “We create a kind of movie that allows us to see a
specific moment in the process where photoluminescence is occurring. Then we
can filter out the shadows that obscure the measurement or spectra we’re
looking for,” he says.
With samples loaded into the spectrometer, researchers yell “Action!” by firing a laser that excites the target. In an edit of the
resulting “movie” (which can be done in real time by the spectrometer), they
chop off the front and back to narrow the data set to a range that might last
only 80-billionths of a second, when the probe signal is strongest and the
background signals are absent.
But it’s critical to know just the right window of time to
look at, Martí says. That’s where the Rice methodology removes any uncertainty.
They let researchers analyze all the factors, such as the emission intensity
and decay of the specific probe with and without the target and the anticipated
level of background noise. The experiment can then maximize the
signal-to-background noise ratio. The technique works even with probes that are
less than optimal, he says.
In combination with a technique called fluorescence lifetime
microscopy, the Rice calculations may improve results from other diagnostic
tools that gather data over time, such as magnetic resonance imaging machines
used by hospitals.
Martí says the equations were the common-sense results of
years of working with fluorescent spectroscopy. But, he says, when he looked
for materials to help teach his students how to use time-resolved techniques to
improve probes’ resolution, he found none.
“I thought there must be some publication out there that
would describe the tools we use, but there weren’t any,” he says. “So we’ve had
to write them.”
To prove their method, Martí and Huang tested ruthenium- and
iridium-based light-switching probes under standard fluorescent and
time-resolved spectroscopy. The hairpin-shaped probes’ middles are designed to
attach to a specific DNA sequence, while the ends are of opposite natures. One
carries the fluorophore (iridium or ruthenium), the other a chemical quencher
that keeps the fluorescence in check until the probe latches onto the DNA. When
that happens, the fluorophore and the quencher are pulled apart and the probe
lights up.
The individual signal is a flash too tiny and quick for the
naked eye to see. “But our instruments can,” Martí says.
“We’re trying to show that you can use time-resolved
spectroscopy for many applications, but to use it in the right way, you have to
do some analysis first,” he says. “If you do it in the correct way, then it’s a
very powerful technique.”
Source: Rice University
