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Imaging live cells in high resolution

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Gary Boas,

Paul D. Ashby wanted a better picture of what is happening in live cells. But Ashby, a researcher in the Imaging and Manipulation of Nanostructures Facility at Lawrence Berkeley National Laboratory, understood the limitations of currently available imaging tools. Optical microscopy has the diffraction limit; electron microscopy requires drying of the cell.

“There’s a real gap there,” he said, noting the lack of access to high-resolution features in live cells. “We just don’t understand what’s happening at the protein assembly level.”

Ashby and colleagues have been developing atomic force microscopy (AFM) to the point where it can address this need. The challenge with AFM is that the amount of force required to perform such measurements is too high. “You end up deforming the cell membrane, and it conforms to your tip,” he said.

Researchers have reported a novel detection scheme that allows use of nanowire cantilevers for atomic force microscopy (AFM). Rather than measure laser light reflected off the back of the cantilever, as is done with conventional AFM, they place the cantilever in the focus of a laser beam and detect the resulting scattering. Thus, they could identify the nanowire’s position. Images courtesy of the Imaging and Manipulation of Nanostructures Facility at Lawrence Berkeley National Laboratory. Illustration by Flavio Robles, Berkeley Lab Public Affairs.

The investigators therefore have been working to reduce the amount of force by confronting the physical limitations that produce that force; namely, the thermal energy created by damping the cantilever. Thermal energy is what produces the force noise inherent to an AFM system, he said. By reducing the size of the cantilever, therefore, you can reduce that noise.

Using nanowires as cantilevers can result in noise an order of magnitude smaller than is possible with conventional cantilevers. Researchers have explored this possibility before, but the idea has not taken hold because they have not had the means to detect the position of the nanowire.

In a recent Physical Review Letters paper, Ashby and postdoctoral fellow Babak Sanii described a novel detection scheme that enables high-sensitivity detection of nanowire deflection and thus, potentially, AFM imaging of high-resolution features in live cells. Here, instead of measuring laser light reflected off the back of the cantilever, they positioned the nanowire cantilever in the focus of a laser beam and detected the resulting scattering, allowing them to pinpoint the nanowire’s position.

Investigators Babak Sanii (left) and Paul D. Ashby are working toward implementation of the nanowires for imaging of live cells. Ultimately, the technique could allow imaging of high-resolution features of these cells. Courtesy of Roy Kaltschmidt, Berkeley Lab Public Affairs.

Further enabling this was the recognition that the nanoscale system would have more forward-scattered than backward-scattered photons, suggesting that they should focus on collecting the forward-scattered photons. “Because people are so used to using a reflected beam to calculate the position of the AFM cantilever, they just hadn’t moved away from [the backward scattering] model,” Ashby said. “By moving to a forward-scattering model, we were able to gather more photons and have a higher sensitivity.”

The researchers tested the novel detection scheme and found that the results were close to what they had calculated theoretically. The sensitivity was a little lower than what they had predicted, but this was probably because of inaccuracies with the nanowires. “The actual nanowires that we have are not the perfect systems that we used as models,” Ashby explained. Ultimately, the experiments validated the potential of nanowire cantilevers in atomic force microscopy.

Next, the investigators plan to test the nanowires for imaging of live cells. They have built an atomic force microscope for use in this study and are testing its imaging capabilities. At the same time, they are pursuing detection mechanisms that are “even more sensitive,” Ashby said, “with even smaller and stiffer cantilevers.”

Jul 2010
Electromagnetic radiation detectable by the eye, ranging in wavelength from about 400 to 750 nm. In photonic applications light can be considered to cover the nonvisible portion of the spectrum which includes the ultraviolet and the infrared.
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