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In a single movie -- a compilation of a million images capturedover about 20 hours -- viewers can see biological structures beginto emerge as a simple cluster of cells morphs into an elongatedbody with tens of thousands of densely packed cells. The movieconcludes when the embryo begins to twitch, driven by contractionsof its newly formed muscles -- moments before the hatching of afruit fly larva just half a millimeter long. The imaging technology -- driven by a high-speed, non-invasivelight microscope that simultaneously captures images from fourdifferent angles -- is described in the June 3, 2012, issue of thejournal Nature Methods . According to its inventor, Janelia Farm fellow Philipp Keller,the new technology will enable quantitative analyses ofdevelopmental processes that were not possible with previousimaging methods. "This is not just a quantitative improvement[over previous technologies]," Keller says. "It is thedifference between being able to quantitatively measure thisprocess and not even being able to follow the cells." Movie 1 . Embryogenesis Simultaneous multi-view imaging captures 19.5 hours of developmentof a single fruit fly embryo. Movie 2. Central Nervous System Development. Simultaneous multi-view imaging of the developing central nervoussystem in a single fruit fly embryo. Movie 3. Cell Lineage Reconstruction Simultaneous multi-view imaging lets users follow the paths ofindividual cells in a developing embryo as they move and divide. Like many developmental biologists, Keller, who works at theinterface of biology, physics, and computer science, wants tounderstand how a single cell transforms into a complex organism."It's a really complex process, and if you want to understandit, I think it is really important that you are able to visualizeand observe it," he says. But conventional light microscopesare too slow to follow the rapid cellular migrations that occur atthis stage of life, and also tend to damage a living embryo withlight before its development is complete. So scientists have beenforced to piece together snapshots of different stages ofdevelopment from multiple organisms, and make assumptions about thechanges that must have occurred in between what they actually see. "This approach is incompatible with the highly dynamic processof development," Keller says. "Snapshots just don'tcapture the essence of it." Furthermore, cells may divide andrearrange themselves differently each time development occurs, socombining observations from multiple organisms could be misleading. To better follow development, Keller says, scientists needed a wayto image at high speeds, over long periods of time, withoutinterfering with the processes they wanted to observe. Frustratedby the limitations of existing technology, Keller set out to designa microscope that would let him investigate the biologicalquestions that fascinated him most. The new microscope, which Keller's team calls the SiMView lightsheet microscope, builds on technology Keller developed withcolleagues at the European Molecular Biology Laboratory inHeidelberg, Germany to address one of the concerns associated withconventional light microscopy: the damaging effects of exposing asample to light. Light is essential to activate and visualize thefluorescent labels researchers attach to molecules inside cells,but too much of it damages cells and causes fluorescence to fade.Keller and his colleagues reduced this damage by illuminating onlya thin section of a sample at a time with a scanned sheet of laserlight, while a detector records the part of the sample being litup. The light sheet technique was fast, and could capture the rapidprocess of development in samples that were not too thick oropaque. In larger or less translucent embryos, however, light fromthe microscope would scatter as it passed through layers of cells,blurring the image. In a fruit fly embryo, for example, only about30 percent would be visible. "You really lose a lot of the information," Keller says."If you want to study cellular dynamics during development toget a complete, dynamic building plan of the entire animal, youcan't afford to not see parts of the sample." The solution was, in concept, simple: build a light sheetmicroscope that collects images from multiple views at the sametime. "Instead of looking at the sample from one direction, welook at it from four directions at the same time," Kellerexplains. In the new microscope, two light sheets illuminate a sample fromopposite directions, and two detectors collect the resultingfluorescence. Each combination of illumination and detection lensprovides a different perspective. Capturing those four views simultaneously eliminates delays causedby rotating a sample repeatedly into new positions -- a traditionalapproach to multi-view imaging -- and makes the microscope veryfast. "We tried to push the speed as much as we could,"Keller says. This version is as much as 50 times faster than lightsheet microscopes that capture multiple views sequentially, hesays. Coordinating the microscope's components to illuminate the sampleand detect fluorescence at the right time and place requirestremendous precision: even fluctuations in room temperature duringthe course of an experiment can shift components out of alignment.Timing is equally important: light sheets that cross paths can bluran image, so they must be staggered by a few milliseconds. Tomaintain precision, the SiMView is equipped with an electronicssystem that makes adjustments in real time. Once Raju Tomer, a researcher in the lab, and Keller hadimplemented the design of the microscope, the team had to confrontanother hurdle: managing and interpreting the tremendous amount ofdata it generated. The default setting for the microscope, Kellersays, is to collect 350 megabytes of data per second. Imaging asingle sample over a full day can produce up to several tens ofterabytes of data -- about the amount of data stored in thecomplete Library of Congress archive. For experiments designed tocompare development under different genetics or conditions, manytimes that amount will need to be stored and analyzed. "Wewant the data," Keller says. "It's not a problem, but itis a challenge." Anticipating that challenge, lab members Fernando Amat and KhaledKhairy devoted their attention to developing computational methodsto identify and track individual cells in the movies produced bythe microscope. Those methods are an essential part of what Kellercalls a complete technology framework for imaging living samples.The team is now collaborating with Janelia Farm colleagues KristinBranson and Eugene Myers to develop new computational strategies toautomate data analysis, Having focused on developing that technology for the past twoyears, Keller says he is looking forward to shifting more of hisattention to biology. He anticipates that his lab will continueadapting the microscope and making its computational processingmore efficient and powerful. "But we now have the technologyto answer most of the questions we are interested in," hesays. Included in the Nature Methods publication is a movie showing the complete development of thenervous system in a fruit fly embryo. Keller explains thatindividual cells can be followed as they give rise to sensoryorgans, brain lobes, and other structures. At the same time, themicroscope offers resolution high enough to zoom in and see thefast dynamic processes at the tip of a neuron's axon. Anticipating future experiments, Keller says the microscope willlet his team not only trace cell lineages in the embryo, but beginto manipulate development to probe the mechanisms that control it.With Janelia colleagues, he has begun to explore otherpossibilities, tracking development in larger, more complexsamples, such as mouse embryos and fruit fly larvae, and monitoringsignaling in the zebrafish brain. "The microscope isn't the limiting factor anymore,"Keller says. "That's really exciting.". The e-commerce company in China offers quality products such as Hair Straighteners Flat Iron Manufacturer , China Straightener Hair Dryer, and more. For more , please visit Hair Straighteners Flat Iron today!
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