- Algorithms Improve Spectral Imaging
GÖTTINGEN, Germany — A team of researchers at the Max Planck Institute for Biophysical Chemistry broke the theoretical minimum for resolution by a focusing light microscope.
More than 100 years ago, Ernst Abbe laid down the law that the wave nature of light limits the smallest resolvable spot size to one-third of a wavelength in diameter, or approximately 200 nm. He predicted that any features smaller than this must always be blurred by diffraction and hence be indistinguishable by focusing light microscopes.
Pyridine nanocrystals were imaged with classical resolution (top) and with subdiffraction resolution (bottom).
Although focusing light microscopes are good for imaging transparent specimens in three dimensions, they were relegated to the sidelines in the race for higher resolution. Electron and scanning probe microscopes took the lead instead.
In the July 15 issue of Optics Letters, researchers Thomas A. Klar and Stefan W. Hell reported that they had resolved a spot that was 30 percent smaller than Abbe's minimum. Using a focusing fluorescence microscope and a pair of laser beams operating at different wavelengths, the team reduced the overall spot size by selectively cooling the fluorescence at the outer rim of the focus. One beam -- a frequency-doubled UV-pulse -- caused excited molecules to be distributed by the fluorophore, while the other, slightly red-shifted fundamental beam quenched them through stimulated emission. Stimulated emission typically amplifies a beam, but in this case it forced the molecules to the ground state by carrying away their energy. By offsetting the two pulses, the team could sculpt the spot size.
The researchers generated the two pulses with a Mira 900 Ti:sapphire laser, pumped by an Innova 400 argon-ion laser, both from Coherent Inc. of Santa Clara, Calif. The Ti:sapphire operated at a wavelength of 766 nm and a 120-fs pulse length. The outgoing beam was divided into an excitation beam and a fundamental. While the pulses of the first beam were frequency-doubled with a nonlinear optical crystal made by Fuzhou, China-based Casix Inc., the pulses of the fundamental were stretched to 40 ps by a Coherent grating compressor/decompressor.
The technique requires pulsed lasers because, to eliminate fluorescence efficiently, the fundamental pulse must do its job in a much shorter time -- a few picoseconds --than the fluorescence lifetime of the dye -- a few nanoseconds.
The researchers said this approach should be applicable to three-dimensional imaging of any live specimen. They also anticipate uses in subdiffraction resolution pump-probe spectroscopy and three-dimensional photochemistry, especially as resolution improves and the range of suitable dyes increases.
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