Glowing nanopillars light up cells

STANFORD (US) — Scientists have created glowing nanopillars that are literally shining light on living cells.

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

To comprehend the potential of using backlighting, it is helpful to understand the challenges to earlier forms of molecular imaging, which shine light directly on the subject area, Cui says.

Scientists hoping for better, smaller molecular imaging have for years been hampered by a physical limitation on the observation volume—how small an area that can be focused on.

The minimum observation volume has long been limited to the wavelength of visible light, about 400 nanometers. Individual molecules, even long proteins common in biology and medicine, are much smaller.

Cui and colleagues were able to use quartz nanopillars that glow just enough to provide light to see by but weak enough to punch below the 400-nanometer barrier.

The field of light surrounding the glowing nanopillars—known as the “evanescence wave”—dies out within about 150 nanometers of the pillar and Voilà: a light source smaller than the wavelength of light and an observation volume that is one-tenth the size of previous methods.

Details of the research are reported in the journal Proceedings of the National Academy of Sciences.

The Stanford nanopillar imaging technique is particularly promising in cellular studies for several reasons.

First, it is non-invasive and so doesn’t harm the cell that is being observed. For instance, a living neuron can be cultured on the platform and observed over long periods of time.

Second, the nanopillars essentially pin the cells in place, promising for the study of neurons in particular, which tend to move over time due to the repeated firing and relaxation necessary for study.

Lastly, and perhaps most important, by modifying the chemistry on the surface of the nanopillars specific molecules can be attracted and observed. Molecules can be handpicked even within the crowded and complex environment of a human cell.

“We know that proteins and their antibodies attract each other,” says Bianxiao Cui. “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.”

To create the nanopillars, the researchers begin with a sheet of quartz, which they spray with fine dots of gold in a scattershot pattern—Jackson Pollock-style and then 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.

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 cell shape of a GFP transfected cell. (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. (Credit: Stanford University)

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 the diameter of the nanopillars by varying the size of the gold dots. Once the etching process is complete and the pillars are created, they add a layer of platinum to the flat expanse of quartz at the base of the pillars.

A light is then shined from below. 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,” says Yi Cui, associate professor of materials science and engineering at Stanford, “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 says.

“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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