Put a materials scientist and a cellular biophysicist in a room together, and what do you get? There are many possible outcomes, and as reported in a recent paper, one of them is light delivered to the inside of cells with tiny nanopillars.

Bianxiao Cui and Yi Cui, both at Stanford University and only coincidentally with the same name, were college classmates in China some years ago. When they both wound up with faculty positions in different departments at Stanford University, they decided to collaborate. Yi Cui, a materials scientist, was working on nanostructures for batteries and solar cells, and Bianxiao Cui set out to find a way to apply this to her work on imaging single molecules in neurons.

“One of the biggest problems in single-molecule imaging is the fluorescence background,” says Bianxiao Cui, and the researchers thought that nanotechnology could help. Existing methods—total internal reflection fluorescence (TIRF) microscopy, for instance—use an evanescent wave to generate a spatially confined zone of excitation so that only a small fraction of molecules are excited and the fluorescence background is reduced. TIRF, however, has the problem of only restricting the light in one dimension. What the researchers wanted was a way to restrict light to a small volume in three dimensions.

Scanning electron micrographs of a cell on nanopillars. Image courtesy of Bianxiao Cui.

An approach that uses what are called zero-mode waveguides does achieve this. In this approach, very tiny holes are drilled into a layer of metal coated onto glass, holes so small that instead of propagating light that is shone onto the metal, they restrict the light within the hole. “Our technique is in some ways similar to the zero mode waveguides,” says Bianxiao Cui, “but instead of making a hole [in the metal] we have a pillar that is poking out.” The researchers constructed transparent nanopillars 100–150 nanometers in diameter on the metal-coated substrate; these are small enough, again, that light shone onto the substrate mostly does not pass through it. A certain amount of light does propagate through the pillar, however, and generates a three-dimensional evanescent wave around its surface.

“The best thing,” says Bianxiao Cui, “is that this interfaces with cells very nicely. The cells love it.” The result, then, is the possibility for spatially very confined optical access to the interior of the cell. Illumination through nanopillars of a cell that expresses GFP cytosolically, for instance, gives very confined spots of fluorescence only at the pillar locations.

The researchers used simulations to model the decay of the light at the tip and the sides of the nanopillars (the light emerges mostly from the sides). They estimate the observation volume to be 10−16 litres, an order of magnitude smaller than that achieved with two-photon excitation. They have grown cortical neurons, hippocampal neurons, cardiomyocytes and a variety of mammalian cell lines on these pillars, and in all cases the cells seem to thrive. “Because of the small dimensions of the pillar, the cell seems to treat it as some sort of cellular organelle, to recognize those dimensions,” Bianxiao Cui says. The researchers continue to investigate the best dimensions and interpillar distances for various applications.

Although others have in the past used nanopillars to deliver molecules to cells, the data at this stage are not entirely conclusive about whether the pillars are topologically inside or outside the cell (that is, whether they access the cytosol or not). Scanning electron micrographs show that the cells seem to engulf the pillars, but the resolution is insufficient to tell whether or not there is a membrane between the pillar and cytoplasm. This will affect the types of experiments that can be done: for instance, recruiting cytosolic molecules to the pillars will only be possible if they are actually inside the cell.

Irrespective of the topology, the pillars can still be used to optically excite molecules in a very small volume. Bianxiao Cui and her lab members are pursuing experiments in which they use the nanopillars to either locally photo-uncage a neurotransmitter or to excite a small number of photoactivatable fluorescent proteins so that the behavior of single molecules can then be studied as they diffuse to dark areas in the cell.