DVD surface imaged by photon tunnelling microscopy. Credit: NANOPTEK

Photon tunnelling microscopy (PTM) is a unique surface-scanning technology that can produce real-time three-dimensional images at unparalleled resolution of biological samples, including live cells. The basic technology can be used with common optical microscopes, and can achieve better vertical resolution than a scanning electron microscope without the need for a scanning tip.

The system addresses a fundamental problem of optical imaging — the smaller an object is relative to the wavelength of incident light, the larger the angle of the diffracted light. For very small objects, the light is diffracted at such a large angle that it cannot leave the object, and is bound in an 'evanescent field', the intensity of which decays exponentially with distance. To see such a small object, you have to take the lens to within a few hundred nanometres of the object's surface without damaging either the instrument or the sample. And unless you operate in a 'clean room', you will have to deal with dust that is many times larger than this distance.

The technology that enables PTM was developed by John Guerra in his former role as senior principal engineer at Polaroid. In June 2002, Guerra launched his own company, Nanoptek, based in Concord, Massachusetts, to develop and commercialize the technology. The key to Nanoptek's approach is a square of soft, flexible polymer film, 75 mm across and 15–20 µm thick, called a transducer, which is applied over the sample. This is made optically continuous to the near-field lens with a fluid coupling. “This allows you to move around this 75-mm area very quickly without damaging the microscope or the sample,” Guerra explains. The film also covers any dust particles on the sample.

Over the 400-nm vertical range of the evanescent field, the coupling goes from total to zero, creating a grey-scale image in the microscope. A 12-bit CCD camera would give a vertical resolution of 0.1 nm — better than that achievable with scanning electron microscopy (SEM), and equal to that of atomic force microscopy (AFM), with the added advantage of real-time imaging.

“You can get 200-µm fields of view at video rates. You can watch chemotaxis in progress or cells moving,” says Guerra. At about 100 nm, lateral resolution is not as good as with SEM or AFM, but is better than that of confocal or phase-shifted interference microscopy. Nanoptek is also planning to combine PTM with phase-shifting to increase the lateral resolution to 0.1 nm. “That will be the next exciting development,” Guerra says. “You will be able to image viruses and very, very small biological materials.”

T.C.