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It quickly becomes apparent how the Bessel sheet fits into the imaging landscape. Like other fluorescence microscopes, the Bessel sheet detects fluorescently tagged molecules within cells, letting biologists follow specific proteins and watch them interact with one another. The basic technique relies on fluorescent tags that absorb light—typically directed at a sample in a focused laser beam—and rerelease the energy as fluorescence, which the microscope captures to create an image. Up to a point, brighter fluorescence contributes to a clearer image, but there is a trade-off. Too much light exposure can make a cell sick, so living samples don’t survive long under the laser’s glare. What’s more, light destroys fluorophores’ ability to fluoresce, meaning even dead cells can be imaged only a brief time before their signal begins to fade. So, fluorescence microscopy often ends up being an exercise in compromise.
Capturing fluorescence becomes increasingly crucial as new technologies push the limits of both spatial resolution and speed. Higher-resolution microscopes must extract information faster, more frequently, and from ever-shrinking segments of a sample. Ideally, a microscope would glean information from each photon of fluorescence a sample emits. In practice, however, plenty of photons bounce off an illuminated sample and are lost forever.
Betzig and his team designed their new microscope to take better advantage of what they call the “photon budget.” Most microscopes illuminate a sample from the same objective lens that collects light, directing a beam all the way through the sample even though only a single plane is in focus at a time. At high-resolution, where every photon counts, this is a problem because light in out-of-focus regions activates fluorescence and damages cells, with no imaging payoff.
An alternative is to direct a sheet of light through the side of a sample, via a lens that lies perpendicular to the objective that collects the returned light. This approach confines the excitation much closer to the portion of the sample in focus. It’s an old idea—first proposed more than 100 years ago—that has recently been adapted to high-speed imaging of multicellular organisms. Ernst Stelzer, who was at the European Molecular Biology Laboratory in Heidelberg, Germany, and is now at the Frankfurt Institute for Molecular Life Sciences, resurrected the technique, Betzig says, taking advantage of the reduction in light damage to image living samples repeatedly over prolonged periods.
The thick sheets of light that have made Stelzer’s technique a success, however, obscure the tiny structures inside cells—a problem that Betzig’s team addressed with a Bessel beam. At its core, a Bessel beam is an extremely narrow beam of light that can be swept across a sample to create a thin sheet well suited for imaging subcellular structures. The Bessel’s central beam is surrounded by concentric rings of weaker light, however, which cause the same problems as any unwanted light sources do. So Betzig, Planchon, and Gao devised a few tricks to minimize the contaminating light. Rather than sweeping the beam continuously, they pulse the beam rapidly to eliminate out-of-focus blur—a technique also used in super-resolution microscopy called structured illumination. They combined this pulsing with two-photon microscopy, a method in which weakly exposed regions generate very little fluorescence. Together, these tricks add up to a microscope that collects unusually detailed images without damaging living cells.
Gao, Planchon, and Chen help students mount their samples so they can receive the sheet of light created by the Bessel beam at the appropriate angle, then tweak the microscope’s parameters to accommodate variations in thickness, brightness, or background fluorescence. The Bessel beam sweeps quickly through each sample, collecting up to 200 images per second. As the microscope’s computer compiles the two-dimensional images into three-dimensional stacks and the first pictures begin to appear on the monitor, what is astonishing is the extraordinary detail apparent in all three dimensions.
Clare Waterman, director of the MBL physiology course, says she was blown away when she saw the Bessel sheet’s first images of her students’ cells. “I’ve been looking at light microscopy images and 3-D reconstructions for 20 years,” she says. “When you rotate a 3-D reconstruction, you always know what the z-axis is.” That is, even when a microscope produces crisp, detailed images in two dimensions, resolution almost always declines significantly in the third dimension. But the Bessel sheet images were different. “I could not tell what the z-axis was,” Waterman says. The cellular detail was clear from any angle.
The three-dimensional movies of migrating cells, which were grown in a collagen gel so they could move freely in any direction, settled for Waterman what she says had been an unresolved debate in her field. Waterman studies cell motility at the National Heart, Lung, and Blood Institute, and lately, she says, there’s been a big push to find out whether structures thus far observed only in cells squashed flat between a microscope slide and a coverslip are physiologically relevant. But, she acknowledges, “There’s been a reason we’ve been doing stuff on a coverslip for so long: because that’s where optics are good.” Technologies that image in three dimensions lack the spatial resolution to determine, for example, whether migrating cells truly project the long, flat extensions thought to propel their motion. “Well, we could certainly see two-dimensional, flat lamellipods with this [Bessel sheet] system that we could measure the thickness of, in every dimension.”