The new approach rapidly corrects for distortions in transparent, nonscattering tissues at the millimeter scale without exposing them to damaging levels of light, making it well suited to imaging the transparent bodies of zebrafish, an important model organism in biological research, researchers said.
A membrane-labeled subset of neurons in the brain of a living zebrafish embryo is seen in a frame grab from an adaptive optics microscope oerating in two-photon excitation mode. Courtesy of HHMI Janelia Farm Research Campus.
Eric Betzig, a team leader at the Howard Hughes Medical Institute's Janelia Farm Research Campus, postdoctoral fellow Kai Wang, and ! colleagues used the technique to bring into focus the fine, branching structures and subcellular organelles of nerve cells deep in the living brain of a zebrafish. These structures remain blurry and indistinct under the same microscope without adaptive optics.
The technique involves two-photon excitation and takes its cues from laser-induced guide stars used to correct for atmospheric turbulence in astronomy and descanning methods used to average out motion-induced errors in retinal imaging.
The researchers shined their own type of guide star across areas of tissue, and the returning light was analyzed by a wavefront sensor to determine what optical corrections were necessary. The adaptive optics compensated for spatial variation in aberrations and recovered diffraction-limited imaging over large volumes (>240 mm per side) with a 14-ms update rate.
"We combined the descan concept from the ophthalmologists with the laser guide stars of the astronomers, and c! ame up with what amounts to a really good solution for aberrat! ing but nonscattering transparent samples, like the zebrafish," Betzig said.
The research was published in Nature Methods (doi: 10.1038/nmeth.2925)
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