Design Breakthrough Improves Lossy Confocal Microscopes

Daniel J. Dufresne

OXFORD, UK -- Engineers at the University of Oxford have discovered a way to get up to 50 times the light efficiency from confocal microscope systems that currently employ a component called a Nipkow disc. By replacing the Nipkow disc with a redesigned aperture mask and then using electronic image subtraction, the Oxford researchers demonstrated that confocal microscopes can produce real-time images with light efficiencies as high as 50 percent. Confocal devices typically use only 1 percent of available light -- or less.
As a result of these low light efficiencies, confocal microscope systems often have to flood specimens with laser light to obtain sufficient illumination, leading to one of the major problems associated with confocal microscope imaging: photodamage. To address this problem, a team led by Tony Wilson, an Oxford professor of engineering science, designed an aperture mask perforated with densely packed random sequences of pinholes instead of the less densely packed patterned arrays found on Nipkow discs. The mask, which leaves one sector completely open, relays 50 percent of the light passed through it, making it much more transmissive than Nipkow discs, whose perforations yield direct confocal images at the expense of excluding all but a tiny fraction of the illumination.
Publishing its results last fall in the journal Nature, the team said it achieved higher light efficiencies with a revamped aperture mask by relaxing the requirement of obtaining pure confocal images directly. Their modified setup produces a conventional image superimposed on a confocal image. The conventional image, digitally encoded by a charge-coupled device camera, can then be subtracted electronically, leaving only the confocal image.
Rimas Ju(breve)skaitis, a research associate in Oxford's department of engineering science and co-author of the Nature article, said the area of greatest demand for his group's aperture mask will be critical dimension measurement, a kind of metrology that currently requires the use of confocal microscopes configured with Nipkow discs.

Noise problems avoidedP> "We also think it could be a poor man's confocal fluorescence microscope," Ju(breve)skaitis said of the new system design, humorously (and temporarily) dubbed SCAM, for scanning correlation aperture microscope. He said the correlation aperture avoids the signal-to-noise ratio problems with Nipkow discs transmitting in the ultraviolet -- an essential spectral range for fluorescence studies. Moreover, Ju(breve)skaitis said the improved light efficiency achieved with his group's device could allow researchers to replace expensive laser illuminators with more affordable arc lamps in fluorescence applications.
Digital image subtraction is a problem where the image signal is very weak, but Ju(breve)skaitis said the system does work well enough to image fluorescence above a minimum threshold. He said ongoing work in applying the device to weak fluorescence microscopy has yielded encouraging results.
While the researchers' paper asserted that they had achieved a net 25 percent light efficiency with their scanning correlation aperture microscope design, Ju(breve)skaitis said a confocal system modified to detect polarized light could achieve a net 50 percent efficiency. "Our mask is not a panacea, but it is a significant improvement on Nipkow disc devices," he said.