| BERKELEY,
CA – Nanotechnology may be in its infancy, but biologists
may soon use it to watch the inner workings of a living
cell like never before. Scientists at the U.S. Department
of Energy’s Lawrence Berkeley National Laboratory (Berkeley
Lab) have developed a way to sneak nano-sized probes
inside cell nuclei where they can track life’s fundamental
processes, such as DNA repair, for hours on end.
“Our work represents the first
time a biologist can image long-term phenomena within
the nuclei of living cells,” says Fanqing Chen of
Berkeley Lab’s Life Sciences Division, who developed
the technique with Daniele Gerion of Lawrence Livermore
National Laboratory.
Their success lies in specially
prepared crystalline semiconductors composed of a
few hundred or thousand atoms that emit different
colors of light when illuminated by a laser. Because
these fluorescent probes are stable and nontoxic,
they have the ability to remain in a cell’s nucleus
— without harming the cell or fading out — much longer
than conventional fluorescent labels. This could give
biologists a ringside seat to nuclear processes that
span several hours or days, such as DNA replication,
genomic alterations, and cell cycle control. The long-lived
probes may also allow researchers to track the effectiveness
of disease-fighting drugs that target these processes.
“We could determine whether
a drug has arrived where it is supposed to, and if
it is having the desired impact,” says Chen.
The first enduring look into
the secret lives of cell nuclei comes by way of a
strong collaboration between biologists and chemists.
For the past four years, Chen and Gerion have worked
closely with members of the lab of Paul Alivisatos,
a Berkeley Lab chemist in the Materials Sciences Division
and Associate Laboratory Director who helped pioneer
the development of nano-sized crystals of semiconductor
materials. Called quantum dots, these microscopic
crystals have shown promise in such wide-ranging applications
as solar cells, computer design, and biology. In 1998,
for example, Alivisatos developed a way to fashion
inorganic nanocrystals composed of cadmium selenide
and cadmium sulfide into fluorescent probes suitable
for the study of living cells. This technology has
been licensed to the Hayward, California-based Quantum
Dot Corporation for use in biological assays.
More recently, Chen and Gerion
wondered if they could get even closer to the genetic
action by transporting quantum dots inside cell nuclei.
“We took the tool Paul developed
and applied it to a problem faced by biologists every
day — getting inside the nucleus, a desirable target
because the cell’s genetic information resides there,”
says Chen.
First, they had to breach the
nuclear membrane, which has pores that are only about
20 nanometers wide. To fit through these tiny slits,
Chen and Gerion used an especially compact cadmium
selenide/zinc sulfide quantum dot coated with silica.
Next, they stole a trick from a virus’s playbook to
smuggle this nanocrystal past the highly selective
membrane that guards the entrance into the nucleus.
In nature, a virus called SV40 is coated with a protein
that binds to a cell’s nuclear trafficking mechanism,
a ploy that gives the virus an unhindered ride inside
the nucleus. Chen and Gerion obtained a portion of
this protein and attached it to the quantum dot. The
result is a hybrid quantum dot, part biological molecule
and part nano-sized semiconductor, that is small enough
to slide through the nuclear membrane’s pores and
believable enough to slip past the membrane’s barriers.
“We
knew we could get quantum dots inside a cell, but
getting them through the nuclear membrane is very
difficult,” says Chen. “So we learned from the virus.”
|
| So
far, Chen and Gerion have been able to introduce and
retain quantum dots in the nuclei of living cells for
up to a week without harming the cell. In addition,
quantum dots fluoresce for days at a resolution high
enough to detect biological events carried out by single
molecules. In contrast, conventional labels such as
organic fluorescent dyes and green fluorescent proteins
only fluoresce for a few minutes at a high resolution.
These labels are also either toxic to cells or difficult
to construct and manipulate.
In the future, they hope to tailor quantum dots to
track specific chemical reactions inside nuclei, such
as how proteins help repair DNA after irradiation.
They have already visualized the dots’ journey from
the area surrounding the nucleus to inside the nucleus,
a feat that opens the door for real-time observations
of nuclear trafficking mechanisms. They also hope
to target other cellular organelles besides the nucleus,
such as mitochondria and Golgi bodies. And because
quantum dots emit different colors of light based
on their size, they can be used to observe the transfer
of material between cells.
“We can have two different quantum dots in two different
cells, and watch as the cells exchange their mitochondria,”
says Chen, adding that their technique paves the way
for imaging a host of other long-term biological events.
“The toughest part is getting inside the nucleus,
and we have already cleared that hurdle.”
Chen and Gerion’s research was published in the 2004,
Vol. 2, No. 10 issue of Nano Letters.
Berkeley
Lab is a U.S. Department of Energy national laboratory
located in Berkeley, California. It conducts unclassified
scientific research and is managed by the University
of California. Visit our Website at www.lbl.gov.
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