“It wasn’t like I was pining away for this,” Waterman says. “I had accepted the limits of optical microscopy and I’d learned to interpret my data within those limits.” Once she saw what was possible, she says, “I was doing backflips for a week.”

Watching Embryos Develop

The Bessel sheet gets little rest at Woods Hole. A grid on the wall blocks out time for faculty who want to squeeze into gaps between student projects, but unexpected visitors stream in at all hours. University of California, San Diego, developmental biologist Andrew Chisholm, who visits MBL as faculty in the neurobiology course, says so many people were in and out of the tiny room that the rising temperature sickened the C. elegans roundworm embryos whose developing skin he wanted to image. With the confocal microscopes Chisholm typically uses, he says, “you’re constantly balancing imaging quality with viability of the embryos. With a conventional microscope you really have to settle for images that don’t look very good.” But since the Bessel sheet limits phototoxicity by using far less light, the dying worms were puzzling.

Chisholm and the Betzig team considered other factors, and discovered that if they imaged the developing embryos in the morning—after a few idle hours had let the room cool down—they had better success. Working with Planchon, Chisholm assembled a movie that shows skin cells forming, making junctions with one another, and then forming a sheet that spreads out to encase the growing embryo. Chisholm says the movie, which spans eight hours of early development, is “an order of magnitude better than what we can get away with using the last generation [of imaging technology]. It’s just beautiful.”

User Feedback

By the time the weary team of postdocs disassembles the microscope for the journey back to Virginia, they have imaged developing embryos, cells suspended in growth media, the nervous system of whole worms, cells embedded in collagen, dying cells, and dividing cells. Over the weeks, Chen says, they have taken every opportunity “to talk to biology people, to listen to what they are thinking. Because it’s really, really different from us.”

They have heard users’ frustration about the microscope’s unconventional sample holder, which requires a fussy assembly process to secure the sample in a vertical position—practical from an engineering perspective, but cumbersome for users. They know they need to adapt their optics to handle thicker samples, which introduce aberrations in the imaging process. And there is an unrelenting desire, biologists have told them, for even better spatial resolution, at even faster speeds.

None of these requests are a surprise, the microscopists agree. And they are prepared to address them. The group plans to build a third version of the Bessel sheet, so they can continue to tweak the instrument and test out improvements while the original setups collect data. Chen will begin work immediately to add additional Bessel beams to the microscope, which he says will increase imaging speed while reducing phototoxicity. A new postdoc in the Betzig lab, Kai Wang, will introduce adaptive optics into the system, and research scientist Lin Shao, who introduced the current methods of analyzing the data generated by the Bessel microscope, will continue to improve those algorithms. Thomas Planchon, now an associate professor at Delaware State University, plans to develop similar microscopes to look at live multicellular organisms and continue working closely with biologists. Ultimately, Betzig’s group would like to merge the Bessel sheet’s capabilities with the super-resolution of PALM, a project that the Galbraiths—frequent collaborators at Janelia Farm who brought their own custom-built PALM with them to Woods Hole this summer—are urging forward.

The long days at MBL will transition into long days back at Janelia Farm, as the Betzig team continues to improve the Bessel sheet. Because what they really learned at Woods Hole, Gao says, is how urgently biologists await those improvements.

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