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Glowing Nanopillars Light Up Cells

STANFORD, Calif., April 14, 2011 — A novel cellular research platform created at Stanford University uses nanopillars that glow in such a way as to allow a deeper and more precise look into living cells.

The Stanford team — led by chemist Bianxiao Cui and engineer Yi Cui (no relation), with Chong Xie and Lindsey Hanson — reported its work in a recent article in PNAS.

“This novel system of illumination is very precise,” said Bianxiao Cui, assistant professor of chemistry. “The nanopillar structures themselves offer many advantages that make this development particularly promising for the study of human cells.”

Longstanding challenges

To comprehend the potential of this breakthrough, it is helpful to understand the challenges to earlier forms of molecular imaging, which shine light directly on the subject area rather than using backlighting, as in this approach.

Image of the interface of cell (blue) and nanopillar shows cell membranes wrapped around the pillar. (Image: Bianxiao Cui, Stanford University)

Scientists hoping for better, smaller molecular imaging have been handcuffed by a physical limitation on how small an area on which they could focus. The minimum observation volume has long been subject to the diffraction limit, but individual molecules — even long proteins common in biology and medicine — are much smaller than visible light’s typical limit of about 400 nm.

This is where evanescence comes in. The team used quartz nanopillars that glow just enough to provide light to see by, but weak enough to punch below the 400-nm barrier. The field of light surrounding the glowing nanopillars — known as the “evanescence wave” — dies out within about 150 nm of the pillar, thus acting as a light source smaller than the diffraction limit. The researchers estimate that they have shrunk the observation volume to one-tenth the size of previous methods.

Particular promise

The nanopillar imaging technique is particularly promising in cellular studies for several reasons. First, it does not harm the cell that is being observed — a downfall of some earlier technologies. For instance, a living neuron can be cultured on the platform and observed over long periods. Second, the nanopillars essentially pin the cells in place. This is promising for the study of neurons in particular, which tend to move because of the repeated firing and relaxation necessary for study.

A scanning electron microscope image of a cell grown over and interacting with nanopillars. Arrows indicate three nanopillars. (Image: Bianxiao Cui, Stanford University)

Lastly, and perhaps most importantly, the investigators found that, by modifying the chemistry on the surface of the nanopillars, they could attract specific molecules they want to observe. In essence, they can handpick molecules to study, even within the crowded and complex environment of a human cell.

“We know that proteins and their antibodies attract each other,” Bianxiao Cui said. "We coat the pillars with antibodies, and the proteins we want to look at are drawn right to the light source — like prima donnas to the limelight.”

Setting the scene

To create their nanopillars, the researchers begin with a sheet of quartz, which they spray with fine dots of gold in a random pattern. They etch the quartz using a corrosive gas. The gold dots shield the quartz directly below from the etching process, leaving behind tall, thin pillars of quartz.

The researchers can control the height of the nanopillars by adjusting the amount of time the etching gas is in contact with the quartz, and their diameter, by varying the size of the gold dots. Once the etching process is completed and the pillars created, they add a layer of platinum to the flat expanse of quartz at the base of the pillars.

Fluorescence imaging using nanopillar illumination in live cells. (A) White-light imaging reveals the locations of nanopillars. (B) Fluorescence imaging by epi-illumination shows the shape of a cell transfected with green fluorescent protein. (C) Nanopillar illumination excites only those fluorescence molecules that are very close to nanopillars inside the cell, giving rise to fluorescence spots perfectly colocalized with the nanopillars. (Image: Stanford University)

The researchers then shine a light from below their creation. The opaque platinum blocks most of the light, but a small amount travels up through the nanopillars, which glow against the dark field of platinum.

“The nanopillars look a bit like tiny light sabers,” said Yi Cui, associate professor of materials science and engineering, “but they provide just the right amount of light to allow scientists to do some pretty amazing stuff — like looking at individual molecules.”

The team has created an exceptional platform for culturing and observing human cells. The platinum is biologically inert, and the cells grow over and closely adhere to the nanopillars. The glowing spires then meet with fluorescent molecules within the living cell, causing the molecules to glow — providing the researchers just the light they need to peer inside the cells.

“So, not only have we found a way to illuminate volumes one-tenth as small as previous methods — letting us look at smaller and smaller structures — but we can also pick and choose which molecules we want to observe,” Yi Cui said. “This could prove just the sort of transformative technology that researchers in biology, neurology, medicine and other areas need to take the next leap forward in their research.”

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There are just a bunch of methods that go beyond Abbe's diffraction limit and can even go lower than this technique down to some nanometers like STEP, STORM etc.
4/30/2011 2:02:11 AM

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