27 Aug 2008
Researchers have extended the resolution of a wide-field microscope beyond the diffraction limit to image cells in finer detail.
A team of Swiss researchers has increased the lateral resolution of a total internal reflection fluorescence microscope by 2.5 times to 92 nm using a specially designed spatial light modulator (SLM). The group says that its SLM can tune the penetration depth of light incident on the sample without the need for other optical components (Optics Letters 33 1629).
"Such a set-up is a step towards structured total internal reflection fluorescence (TIRF) microscope systems that could be used by non-experts," Andreas Stemmer, a professor at ETH Zurich, told optics.org. "Previous set-ups were too complicated to be operated by average microscopy users. Our method affords flexible and fast adjustment of the illumination parameters, which is important for TIRF experiments and guarantees ease of use."
In TIRF microscopy an evanescent field selectively excites fluorophores adjacent to a coverslip. The resulting optical slice is very thin (<100 nm) and effectively eliminates out-of-focus fluorescence. In contrast, lateral resolution in TIRF microscopy remains diffraction limited.
To overcome this constraint, Stemmer's group uses a sheared (tilted) diffraction grating written onto a SLM to adjust the illumination depth in very fine steps. "Our work is a versatile set up that allows one to tune the illumination parameters," commented Stemmer. "Fine tuning of the incident angle allows the penetration depth of the evanescent field and hence the thickness of the optical section to be controlled."
In the set-up, the SLM is used as a diffractive optical element in a common path interferometer. The sample is illuminated with an evanescent standing wave orientated along the x direction. Three images are recorded and for each one, the phase of the standing wave is changed by pi/2. The evanescent standing wave is rotated by 90° and again three images of varying phase are recorded.
"Interference is generated in the coverslip, which has a higher refractive index than the watery medium, enabling the generation of very fine interference patterns (fringe periods down to 175 nm using light with a wavelength of 488 nm)," explained Stemmer.
In the post-processing step, the raw images are filtered and the high-frequency information is demodulated by solving an equation system in fourier space. "The spectral information that was shifted by the physical convolution upon structured illumination is computationally shifted back to its proper location in fourier space," explained Stemmer. "Finally the reconstructed object spectrum is back-transformed in real space, giving an image of enhanced resolution."
The next step for the team is to apply its technique to answer problems in biology and other scientific areas.