[Paleopsych] Nano-Probes Allow an Inside Look at Cell Nuclei

[Steve Hovland]

 Contact: Dan Krotz, (510) 486-4019, dakrotz at lbl.gov 
<mailto:dakrotz at lbl.gov?subject=Re:%20Nano-Probes%20Allow%20an%20Inside%  
20Look%20at%20Cell%20Nuclei>
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) and Lawrence Livermore National Laboratory 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."
	
 	
	
These two images portray the movement of the nano-sized probes. On the 
left, a false-color overlay of fluorescence from a cell taken at four 
minute intervals reveals the dots moving from the green to the red 
positions. On the right, a large aggregate of immobile dots is indicated 
with the red arrow, while the circled stars and arrows indicate dots that 
move. 	
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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