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Joining Team EMBL
By 2005, Keller was working on his doctorate, becoming interested in studying life at its inception, and had already joined a team of scientists at EMBL led by Ernst Stelzer. The group had developed a new type of microscope for observing the previously invisible processes of embryonic development.
The scientists used a relatively simple trick from 100 years back. In 1903, chemist Richard Zsigmondy had invented the “ultramicroscope,” which illuminated a sample through a slit at a right angle to the viewing angle. Using the same principle, the EMBL researchers engineered a more sophisticated version for the 21st century, taking full advantage of the modern computing power needed to process and analyze massive amounts of data. They published the innovation in 2004 in Science, calling it selective plane illumination microscopy, or SPIM.
“Instead of collecting data point by point, using the same objective for illumination and fluorescence detection as the confocal microscope, we illuminate an entire plane [of the specimen] from the side,” Keller says. The only thing you have to do is collect the fluorescence emitted at a right angle from this plane using a camera with a conventional detection system. It’s relatively simple.”
The inaugural version of the microscope yielded the first video of an embryo’s beating heart—that of a Medaka, or Japanese rice fish. After refining the instrument for version 2.0—dubbed “digital scanned laser light-sheet fluorescence microscopy,” or DSLM—Keller and his EMBL colleagues captured the first 24 hours of a zebrafish embryo’s development with unprecedented resolution and speed. They published their results in Science in 2008.
Progressing beyond day one of development became the challenge. As the embryo grew more complex, images blurred. The increasingly complicated and numerous structures in the rapidly developing fish scattered the light from the scope. To maintain high resolution of these complex images, Keller and his colleagues tweaked the scope so they had rapid electronic control over the pattern of light passing through the specimen. Increased control meant the instrument could accommodate the denser embryos of other lab animals, such as fruit flies and mice.
The scientists took nearly one million images over three days to follow neural development of a zebrafish embryo into its juvenile stage. They also created a “digital fly embryo,” a three-dimensional reconstruction of early Drosophila development with single-cell resolution. The group published their results in the August 2010 issue of Nature Methods.