Many of the complex organic dyes, or labels, that biologists and biochemists use to track molecules inside cells were invented in Germany over 100 years ago. As a result, dye chemists such as Luke Lavis sometimes have to refer back to the original German-language manuscripts for guidance. Lavis doesn't speak German, so he looks to his wife and her Swiss parents for help. None are scientists, but aside from technical words he looks up in a specialized dictionary, “my mother-in-law is very good” at translating, he says.
However, Lavis points out that even translated, the papers represent chemistry a century old. One project Lavis will undertake while at Janelia Farm is to use modern methods to revamp some of the techniques. “There are more and more chemical reactions available,” says Lavis. “Can we apply those new chemistries to fashioning dyes?”
As with the other projects Lavis will undertake, the goal in revamping dye chemistry is to improve the ability of biologists and biochemists to tackle complex questions. “I don't want to do chemistry and stop there,” he says. “The point is, we're going to make better tools.”
Lavis, who calls himself a chemical biologist, has been designing and fabricating dyes since he graduated with a degree in chemistry from Oregon State University in 2000. As an undergraduate, he worked in a natural products chemistry lab, recreating complex organic molecules from tree sap and red wine. Lavis loved it, but was also frustrated by the lack of applications. The natural products synthesis he was doing, he says, was to some extent an academic exercise. The mentality was, “I'm going to make this molecule because it's there.” The usefulness of the molecule was secondary.
Uncertain about whether to pursue a research career or go to medical school, he took a break after graduation by going to work for Molecular Probes, a chemical maker based in Eugene, Oregon. Now owned by Invitrogen, Molecular Probes was, at that time, focused on making fluorescent dyes. While he was there, Lavis got a chance to make molecules with real-world uses. “It was very gratifying to make something, give it to a biochemist or biologist, and have them say `Wow; this really works well, and we can now answer some biological questions.'”
Lavis only spent about eight months at Molecular Probes before his supervisor moved to Molecular Devices, in Sunnyvale, California, and convinced Lavis to join him. At Molecular Devices, Lavis again worked on fluorescent dyes, creating kits that scientists could use to measure such things as ion concentration in cells and voltage differences across cell membranes.
Lavis spent three years at Molecular Devices, enough time to decide that research, not medicine, was the path for him. He joined a chemical biology group at the University of Wisconsin in Madison as a Ph.D. student.
There, Lavis worked in the lab of Ronald Raines, doing basic research on an enzyme called ribonuclease A. Variants of RNase A can selectively kill cancer cells, and Raines's group was trying to figure out how it worked. “In order to do that, we had to develop tools to look at the trafficking—how does it get in [to the cell], where does it go, all those sorts of things,” says Lavis. He developed a fluorescent dye that could be attached to ribonuclease A variants, but which remained dark outside the cell. When the protein-dye pair entered the cell, natural cellular enzymes called esterases automatically snipped a bit of the dye off and made the molecule fluoresce. “You could have a lot of protein outside the cell that remained invisible, and only the molecules that made it inside would light up,” says Lavis. “That allowed us to do a lot of really nice experiments, and those experiments are continuing.”
Toward the end of his time at the University of Wisconsin, Lavis heard about Janelia Farm from another graduate student. He was attracted to the highly collaborative, focused atmosphere—similar to what he had encountered in industry and in the chemical biology group in Madison. “I decided to go for it,” says Lavis, because of the twin focuses of high-end imaging and neuroscience. “If I want to make [labels] to answer biological questions, I want to be near biologists.” But, he says, he also wants to be near the microscope makers that work with the biologists. At Janelia, Lavis can get both.
Along with his effort to modernize dye chemistry, at Janelia Lavis will work with group leader Eric Betzig and fellow Harald Hess to develop new fluorescent tags for their PALM (photoactivated localization microscopy) system. PALM obtains super-high-resolution images by shining low levels of a specific frequency of light at special proteins that fluoresce in response.
The photoactivated fluorescent proteins have only been developed in recent years, and according to Lavis, they have revolutionized imaging techniques. But, he says, the repertoire of such large, complex proteins is limited, restricting the range of processes they can be used to examine. Lavis thinks that more traditional “small-molecule” labels, such as the ones he attached to ribonuclease A during his Ph.D. research, still have a place as complements to the newer techniques. Lavis will work to develop small-molecule tags for use with PALM and similar systems.
Lavis's third project will focus on improving the ability of small molecule fluorescent-ion indicators to operate in living tissue. Ions such as calcium have key roles in the cell, and visualizing their concentrations is important for cell biologists, but he notes that “a lot of the fluorescent indicators we have were designed to work in cultured cells. … Scientists want to use them in live animals.” Lavis says he'll be working on better ways to deliver the compounds into cells, and also—again extending his Ph.D. work—see if he can devise ways to use cells' own enzymes to unmask his tags, in the same way he used them to unmask his dyes bound to ribonucleases.
Lavis envisions collaboration with many groups at Janelia to explore further refinements of this strategy. He wants to see if he can create fluorescent indicators that would be unmasked only in certain cells within the brain of a mouse. “We could look at [the linkage between calcium concentration and neuron activity] in just that small population of cells, rather than the whole thing.”
“With the audacious goals of Janelia, it's going to take very collaborative research. Along with all the fantastic biologists asking the biological questions, we will require tool developers to develop state-of-the-art, next-generation tools so we can do the experiments we need.”
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