Janelia scientists have borrowed a technique from the field of astronomy to overcome biology’s equivalent of the twinkling of stars and the shimmering mirages in desert landscapes.

When it comes to microscopy, blur is bad. A crisp image can tip the evidentiary scale toward one of several competing theories about the identity of a cellular or tissue feature, how it is structured, or how it works. A blurry image? It’s just as likely to fuel speculation and debate, or even deceive researchers into seeing things that are not there. Ever since Antoinie van Leeuwenhoek and Robert Hooke launched microscopy onto the scientific stage in the 17th century, reducing blur has been a relentless theme of microscope developers.

Eric Betzig, a group leader and self-proclaimed “gizmologist” at the Howard Hughes Medical Institute’s Janelia Farm Research Campus, has been adopting that pursuit as his own. In his latest attack on the limitations of modern microscopy, he has borrowed a technique called adaptive optics from the field of astronomy. Astronomers have used this strategy for decades to offset the atmosphere’s capacity to blur light coming into ground-based telescopes from the cosmos.

What Betzig, Janelia Farm colleague Na Ji, and Daniel E. Milkie, an expert in instrumental control systems with Coleman Technologies, Inc., in Chadds Ford, Penn., aim to overcome is biology’s equivalent of the twinkling of stars and the shimmering mirages in desert landscapes. These visually distorting optical effects are due to atmospheric inconsistencies that distort the pathway of light. As a result, starlight appears unruly and a distant mountain range in the desert can look more liquid than solid.

“It’s the same problem inside of cells,” Betzig says. “When light rays move through specimens with organelles, blood vessels, and other structures, the rays are bent.” When viewing isolated cells, this is not so much of a problem. But for thicker multicellular structures, like the cortex of a brain, these heterogeneities prevent light rays traveling through different parts of the specimen from converging into as fine a focus as they would were the specimen homogeneous like glass. This problem has limited biologists’ ability to examine many structures in their intact state—even though their functions are dependent on their three-dimensional architecture and interconnections.

This is where Betzig, Ji, and Milkie have played the adaptive optics card. In astronomy, researchers use an upwardly-directed reference laser to create an “artificial star.” They use this laser-made point of light to measure light-distorting features in the atmosphere. They then use those measurements to sculpt a deformable telescope mirror into a complex shape that cancels out the atmospheric aberrations. The payoff: ground-based telescopes that can view the cosmos almost as though they were above the blurring influence of the atmosphere.

In the journal Nature Methods, in an article published online on December 27, 2009, Betzig and his colleagues describe how they applied adaptive optics techniques to devise high-resolution microscopy that they hope will open new windows on biological structures as pivotal and complex as the thickets of cells in the brain.

Central to the technique is a liquid crystal “spatial light modulator,” which both measures and samples’ optical variations and then sculpts a wave of light into a shape that all but nullifies the sample’s own image-blurring inconsistencies.

With control algorithms devised by the researchers, the liquid crystal element specifies a sequence of illumination patterns that serially probe the deflections of incoming light rays in tens or even hundreds of specific regions of the sample by measuring the image displacements caused by such deflections. An algorithm then translates these measurements into control signals that transform the same liquid crystal component into a mask that tilts the light rays so they converge at a common point, thus negating the sample’s own optical aberrations.

So far the researchers have proven the principle by successfully imaging one-micron diameter spheres tucked underneath a 300-micron thick slice of mouse brain tissue and neurons up to 400-microns deep inside mouse brain tissue. Despite the tissue’s textural complexity, the minuscule test sphere showed up about as sharply as is theoretically possible, Betzig says.

One eventual goal, the researchers say, is to hone the technique so that investigators can image through the full depth of an animal brain’s cortex even as the animal is awake and responding to stimuli. This would help fill an existing observational gap, and allow structural and functional imaging of all the interconnected cells that define the neural circuits of the cortex, rather than just those in the superficial layers.

In talks on her tool-development at Janelia, Ji draws from the preface of Hooke’s pioneering microscopy tome Micrographia, published in 1665. Central to Hooke’s view of science, Ji tells her audiences, is, in his words, “a watchfulness over the failings and an inlargement of the dominion, of the senses.” Identifying where scientific tools are falling short of what they can be and finding ways of enlarging their value, says Betzig, is how he and his colleagues are following Hooke’s lead.

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