The big challenge for the biologists is that many cellular structures and processes fall just below the physical diffraction limit of traditional non-destructive imaging techniques. Fluorescence microscopy, for instance, offers a resolution of 200 nm at best. The physicists' task, therefore, is to break through this limit and develop new optical imaging techniques that meet current biological objectives. And that's exactly what's happened of late, with several novel ways of enhancing the resolution of fluorescence microscopy emerging in recent years.

The authors of the Physics World article - William Barnes, a professor of photonics at the University of Exeter in the UK, biophysicist Paul O'Shea and optical engineer Michael Somekh, both at the University of Nottingham, UK - highlight three fluorescence microscopy-based techniques that show promise for what they call "super-resolution" imaging: stimulated emission and depletion (STED) microscopy, stochastic reconstruction microscopy (STORM) and structured illumination microscopy (SIM).

STED, for example, works by illuminating a sample with a diffraction-limited spot while a second, doughnut-shaped beam simultaneously de-excites fluorophores around the spot's edge. This results in the image being formed only from photons emitted in the smaller central region. In the latest issue of Nature Methods, researchers from the Max Planck Institute for Biophysical Chemistry, Germany, report the use of STED-based imaging to view the inside of a cell with a resolution of 40-45 nm (Nature Methods 5 539). The researchers - led by physicist Stefan Hell - came up with a scheme dubbed isoSTED that combines elements of STED and 4Pi microscopy (in which a pair of constructively-interfering laser pulses illuminate the sample). "We have produced the smallest focal spots attained so far in a scanning fluorescent microscope, and thus attained the best 3D resolution inside a transparent object such as a cell," claimed Hell.

The second technique picked out by the Physics World authors, STORM (also known as photo-activated localization microscopy), exploits the fact that individual objects can be located with much greater precision than two points can be resolved. STORM uses switchable fluorophores that are all rendered dark by a laser beam, and then switched on at random by a brief pulse from a second laser. The process is repeated until all of the fluorophores have been lit up and located, and a full image reconstructed. This technique boasts a resolution as good as 20 nm, although the time required to run all the imaging cycles limits its use in live cells.

While STED and STORM boast impressive resolution, they both require quite complex equipment. This is not the case for SIM, which is claimed to achieve super-resolution with relatively small modifications to a standard full-field microscope. SIM works by shining patterned light onto a structured sample, producing a set of low-resolution Moiré fringes. By recording several Moiré images at different orientations and applying some simple maths, a high-resolution image of the original pattern can be reconstructed. Illuminating a sample with such "structured" light can double the resolution of a conventional microscope.

This week's issue of Science features the extension of SIM to 3D imaging, in which three beams of interfering light generate patterns in the x, y and z directions. This 3D technique could image cultured mouse-tissue cells at a resolution approaching 100 nm - double that of a conventional microscope. Three-dimensional SIM enabled viewing of several features that previously were only seen using electron microscopy (Science 320 1332). While the resolution achieved here is less than that afforded by STED or STORM, the authors claim that "3D-SIM is currently the only subdiffraction-resolution imaging technique that can produce multicolour 3D images of whole cells with enhancement of resolution in both lateral and axial directions."

As pointed out by Barnes, O'Shea and Somekh, it does indeed appear that the experimental needs of biology are driving rapid developments in imaging technology. "We are seeing a change in the nature of biological investigation as it takes on a sounder theoretical basis coupled to experimental analysis - the hallmarks of modern physics," they concluded in their Physics World article. "These are exciting and interesting times to be working in biological research - and not just for biologists."