Microscopy
| A new imaging platform called a “lattice light sheet” is a significant leap forward for light microscopy. It captures high-resolution images rapidly and minimizes damage to cells, so it can image the three-dimensional activity of molecules, cells, and embryos in fine detail over longer periods than was previously possible. |
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Powerful new microscopes have dramatically sharpened biologists' focus on the molecules that animate and propel life. Now, a new imaging platform developed by Eric Betzig and colleagues at the Howard Hughes Medical Institute's Janelia Research Campus offers another leap forward for light microscopy. The new technology collects high-resolution images rapidly and minimizes damage to cells, meaning it can image the three-dimensional activity of molecules, cells, and embryos in fine detail over longer periods than was previously possible.
The developers of the new lattice light sheet microscope have teamed with cell and molecular biologists to produce stunning videos of biological processes across a range of sizes and time scales, from the movements of individual proteins to the development of entire animal embryos. The scientists, including postdoctoral researchers Bi-Chang Chen (now at the Research Center for Applied Sciences in Taiwan), Wesley Legant, and Kai Wang, described their new technology and its applications in the journal Science.
Betzig, one of three scientists sharing the 2014 Nobel Prize in Chemistry, has developed a suite of new imaging technologies over the last decade. The techniques have improved biologists' ability to visually track the movements of cells' tiniest structures – but there were always trade-offs. Imaging cells at high resolution in three dimensions usually meant sacrificing imaging speed, as well as subjecting cells to significant light-induced toxicity.
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“What happens is you end up designing the questions you ask around the tools that are available,” Legant says. With the lattice light sheet, the Betzig team can now optimize their imaging technology for the questions that biologists want to answer.
The new microscope evolved from one Betzig unveiled in 2011. The Bessel beam plane illumination microscope illuminates samples with a virtual sheet of light, created when a beam of non-diffracting light called a Bessel beam sweeps across the imaging field. It produces high-resolution images with less light damage than a traditional microscope, and is fast enough to record dynamic processes in living cells.
Betzig's team had to incorporate a few tricks into that microscope to address a problem that arose from the shape of the Bessel beam. “It's not just a thin pencil of light – it has these dimmer side lobes,” Betzig explains. “So when you sweep that across a sample, you get out-of-focus light.” One solution was to shift the beam step-by-step across the sample, illuminating it with a grating pattern. The researchers could then computationally strip out the blurriness caused by the Bessel beam's side lobes – a technique known as structured illumination.
Structured illumination can also be used to overcome light microscopes' usual limits of spatial resolution. To apply a super-resolution structured illumination technique developed at Janelia by the late Mats Gustafsson, Betzig's team moved the Bessel beam to produce a lattice-like pattern of light. “With that we not only get rid of the side lobe stuff, we actually push the resolution a bit beyond the diffraction limit,” he says.
To reduce the time required to move the Bessel beam each time a sample was imaged, the developers split the beam into seven parallel parts, so each traveled just one-seventh of the original distance. Suddenly, the cells they were imaging seemed healthier. “What was shocking to us was that by spreading the energy out across seven beams instead of one, the phototoxicity went way down,” Betzig says. “What I learned from that experience is that while the total dose of light you put on the cell is important, what's far more important is the instantaneous power that you put on the cell.”
Could they reduce light toxicity even more by dividing the Bessel beam? Betzig wasn't sure what would happen when the beams began to crowd one another, so he modeled the potential effects. Although the beams interfered with one another, the model proposed certain arrangements where that interference actually destroyed the undesirable lobes of light. If he could reproduce those “magic periods” with actual light, the imaging potential was huge. “What you get is sort of a triple win,” he says. “You spread the energy out, this highly structured plane is ideal for doing structured illumination with high contrast, and you get rid of that nasty side lobe problem.”
That's when Betzig dusted off an idea he'd had more than 10 years ago. Working theoretically, he'd proposed a technique he called optical lattice microscopy, which would limit damage to cells by illuminating a sample with a massive three-dimensional array of light foci. Other technologies had taken hold when Betzig moved to Janelia in 2005, and he'd never built an optical lattice microscope. But the idea remained sound. “We used the lattice theory to predict the patterns that would produce these magic periods,” Betzig says.
"We know what the microscope can offer in terms of the imaging, but I think there are a lot of applications we haven't even thought of yet." |
Thirty teams of biologists have come to Janelia over the past year to find out what the lattice light sheet microscope can reveal about the systems they study. Chen, Legant, and Wang have worked with the researchers to optimize the technology for a variety of experiments. “This is not a single imaging technique,” Wang says. “It's an imaging platform.”
The Science paper includes videos of these and other processes gleaned from the Betzig team's collaborations, but the scientists say they represent only the beginning. “We know what the microscope can offer in terms of the imaging, but I think there are a lot of applications we haven't even thought of yet,” Legant says.
Betzig's team freely shares its designs, providing detailed instructions to scientists with the expertise to build their own version of the instrument. Zeiss has licensed the Bessel beam and lattice light sheet microscopy. “It takes a huge amount of effort to move from a successful high-tech prototype to broader adoption of an imaging technology,” Betzig says. “Ultimately, commercialization is the crucial last step to ensuring that these technologies can have broad impact in the research community.”
SOURCE Howard Hughes Medical Institute
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