Blurry vision belongs to history

The classical view that lens-based optical microscopes cannot resolve details separated by less than half the wavelength of light is increasingly becoming a dated one. Several microscopy techniques, used either alone or in combination, can now beat the diffraction limit by at least a factor of 2. Now, in a paper appearing in Physical Review Letters, Claus Müller and Jörg Enderlein at Georg August University in Göttingen, Germany, add to this arsenal of techniques a method that essentially involves modifying an existing bench-top confocal-laser scanning microscope [1]. This type of microscope—called a CLSM for short—is already a fundamental tool for research, particularly in the biological sciences, and Müller and Enderlein’s proposal could influence a broad community. Müller and Enderlein’s technique is built on the method of structured illumination microscopy [2]. When two fine patterns—like that of two combs with different teeth spacing—superimpose multiplicatively, they create moiré fringes (Fig. 1, left). In the case of structured illumination, one pattern is the fluorescence of an unknown sample, while the other pattern comes from the “structured illumination” source, which has a known spatial dependence. The product of the sample and the illumination patterns generates an image that has spatial frequencies that are both sum and difference frequencies between those contained in the original patterns. The difference frequencies, which make up the moiré fringes, have a lower spatial frequency than either of the original patterns and it is these fringes that can often be resolved by a microscope, even if the spatial frequencies of the original patterns themselves are too high to be resolved (see the sequence of panels on the right of Fig. 1). To extract the unknown sample pattern, one needs to do the Fourier back-transformation from frequency space to real space, including a “filter function” that FIG. 1: The basis of structured illumination. (Left) Moiré fringes (dark bands) form between two periodic patterns. (Right) The blue circle (I) indicates the “observable region” of spatial frequencies in a conventional microscope. The three Fourier components of a sinusoidally striped illumination pattern (blue dots) must fall within this circle to be observable within the diffraction limit. (II) Illuminating the sample with structured light extends the observable region in (I) to contain the spatial frequencies within two offset regions (violet). (III) Moving and rotating the structured illumination can extend the frequency space by as much as a factor of 2. (Illustration: Alan Stonebraker)