Lillian Fritz-Laylin is observing a strain of leukemia cell that zips along at about 10 to 20 microns per minute. She’s looking for the motive secret of how these speed demons of the cellular world get around, and she’s doing it by making a high-resolution 3-D micro movie.
“People have studied for a long time how slow cells move…and [they] have largely assumed that fast cells use the same mechanism—just faster,” says Fritz-Laylin, a postdoc scholar at the University of California, San Francisco. But there is no direct evidence to confirm that assumption. So she wants to see exactly how these speedsters get around.
When Fritz-Laylin was interviewed she was at the Howard Hughes Medical Institute’s Janelia Farm Research Campus, working in the lab of Eric Betzig—a trained physicist now specializing in developing cellular imaging technologies. She was making use of the newest version of a breakthrough technique that Betzig unveiled in 2011, called Bessel beam plane illumination microscopy. The results are stunning high-resolution, 3-D movies of cells that look like the creation of a computer animator, not a physicist. The films make the cells look like tiny, cognizant animals, rather than bumbling sacks of fluid.
AFRICAN GREEN MONKEY CELL: An African green monkey kidney cell expressing the gene c-src, imaged using Bessel beam plane illumination. Membrane ruffles surround and engulf extracellular fluid (top panels), creating the spherical intracellular vacuoles seen inside the cell (bottom panels). Credit: Liang Gao and Eric Betzig, Janelia Farm
Fritz-Laylin will not only clock the speed of the cell (which can be done with lower resolution techniques); she’ll look at the way the cell deforms itself as it moves as well as how the flow of its internal structures contributes to the movement. Looking very far down the road she says it is possible scientists might build nano devices based on natural cellular motion that could deliver drugs to specific locations in the body.
The Bessel beam technique, however, is not the only game in town for 3-D movies of cells, and it is not the highest resolution. But Betzig says that in terms of combined spatial resolution and temporal resolution, there is no technique like it. He feels he has landed in an area with the right balance of variables, or, as he calls it, “a sweet spot of spatial and temporal resolution.”
“A brilliantly simple idea”
The basic setup of the new technique involves a sheet of light that sweeps across the cell, illuminating thin slices of its body (inside and out, as the cells are largely transparent), like a harmless deli slicer. A camera pointed perpendicular to the sheet captures images of the 2-D slices, which are eventually stacked together into a 3-D image. The sheet of light scans the body of the cell in two different laser colors in about one second, during which time about 200 images are taken. The machine can repeat this scanning motion for many minutes, and it scans quickly enough to follow the motions of live cells in a free-range environment.
With the Bessel beam technique only the camera’s plane of focus is illuminated. This differs from most imaging techniques where the entire sample is bathed in light. In a traditional microscope for example, the sample is lit with an all-encompassing beam from above. In confocal microscopy the focal plane is imaged with a single, intense point of light, centered between two cones of light that flood the rest of the sample. The excess light doesn’t help image the sample; in fact, all that bright light may quickly burn the cell and kill it.
The “brilliantly simple” idea, as Betzig calls it, of using a sheet of light to reduce cell damage is called plane illumination microscopy. It was first theorized by Austro-Hungarian chemist and 1925 chemistry Nobel Prize winner Richard Adolf Zsigmondy along with German physicist Heinrich Siedentopf in 1903. It was then largely forgotten until the 1990’s when scientists in microscopy started toying with idea of using it again. In 2004 Jan Huisken of the Max Planck Institute of Molecular Cell Biology and Genetics and colleagues published a paper establishing structured plane illumination microscopy, or SPIM, and the field has been booming ever since.
SPIM is useful for imaging multicellular structures, but dropping down to single cells presents a challenge: The method for creating the sheet of light is limited, such that the thinner the sheet gets, the narrow it gets. At a sufficient thickness for imaging cells, the light is reduced to a small point rather than a wide sheet. So SPIM sheets are often only slightly thinner than the individual cells; being able to slice the cell up into thinner sheets would mean better resolution.
So to create a wide, thin sheet of light, Betzig uses something called a Bessel beam. If you’ve ever used a magnifying glass to start a fire, you know that sending light through a lens can focus it into a point. The lens actually creates a funnel of light, with the photons all coming together at the neck. A Bessel beam uses the same idea, but light only enters the lens around the outer edge. Rather than creating a funnel of light with a flat focus point, a Bessel beam creates a long, thin beam of focused light. Bar-code scanners use Bessel beams because the pencil-like beam of light they produce is thin enough to read between the lines of a bar code; even so, scanner lasers are much thicker than the ones used by Betzig.
The most recent incarnation of Betzig’s technique positions seven Bessel beams next to each other to create a “sheet.” Bessel-beam aficionados will know that in addition to the central pencil of light, a Bessel beam also creates rings of subsequently dimmer and dimmer light. The whole thing looks like a bull’s-eye that gradually fades out. Those rings would make an image blurry, but Betzig and his team have found a way to use destructive coherence to get rid of the rings all together.
So the Bessel beam can scan a cell with the same total flux as the light spots used in confocal microscopy, but that flux is distributed among the seven beams and the cell isn’t flooded with excess light; thus, the cell isn’t damaged nearly as quickly. Over time even the Bessel would start to degrade it, but so far the team can watch single cells for hours if necessary.
Just around the corner from where Fritz-Laylin is doing her research, Betzig has another instrument available to visiting users. This one is called structured illumination microscopy, SIM, and it falls under the heading of “super resolution.” Although SIM and other superresolution techniques are pushing to spatial resolutions as high as 20 nanometers, they do not have the field of view of a technique like Bessel beam microscopy; they can only see a very small area, and imaging a larger area with those techniques takes a long time. Most of the techniques therefore require that the cell be stationary or dead. The Bessel beam technique strikes a different balance of variables: imaging live cells, at that sweet spot of spatial and temporal resolution.
That’s not to say that either technique is necessarily better than the other. Scott Fraser, a cell biologist at U.C. San Diego, uses the analogy of watching a football game: a technique like Bessel beam illumination is like a wide-view of the whole field, whereas super-resolution techniques are like binoculars. “You wouldn’t want to watch the whole game through binoculars,” Fraser says, because of course you wouldn’t be able to see what was actually happening in the game. “But maybe during a time out or a huddle, when you want to see the quarterback’s face, you switch to the binoculars.” In fact, cell biologists require an entire toolbox of imaging techniques in order to get the answers they want. It’s unlikely any single technique will ever serve every purpose.
Molecular motion pictures
“I would say this technology is kind of a dream come true for us,” said Chris Janetopoulos, who leads a cellular biology research group at Vanderbilt University. Janetopoulos and his group study Dictyostelium discoideum cells, which are technically single-cell organisms, but have the remarkable ability to join together into a single organism when times are tough, and migrate to a location where food is more plentiful.
HEALTHY DICHTY CELL: A plump, well-nourished Dictyostelium discoideum cell uses ruffles in its membrane to bring in extracellular fluid, which contains chemical information about its environment. Credit: Xufeng Wu, NHLBI; Liang Gao and Eric Betzig, Janelia Farm
STARVING DICHTY CELL: A starving Dictyostelium discoideum cell changes its physicality when food is scarce: it reaches out for other “dichty” cells to join with. These “social amoeba” may come together and travel as a multicellular organism to find food. Credit: Chris Janetopoulos, Vanderbilt; Bi-Chang Chen and Eric Betzig, Janelia Farm
Janetopoulos wants to know exactly how the “dichty” cells change physically as their environment changes. Research suggests that very scarce chemical cues can trigger physical changes in the cell, including remodeling the plasma membrane and recruiting cytoskeletal machinery, to transform the dichty from a fat, happy cell to an elongated cell with a distinct front and rear end, ready to migrate. The morphological changes that dichty’s undergo also occurs in many of the cells of the human body, including a type of white blood cell called a neutrophil. These are some of the fastest migrating cells in the human body, and they are the first responders at damaged locations or areas of infection and begin the process of healing.
With the Bessel beam technique, the group can image D. discoideum long enough to observe the cell’s motion, which might take place over an hour, but also with a high frame rate to capture rapid molecular changes in just a few seconds. “I think back to when I was a graduate student in the late 1990’s. We were just being able to do 3-D reconstructions,” Janetopoulos says. “We could image things pretty well by taking z sections of fixed (dead), immobile specimens. But one of my dreams at the time was to do 3-D imaging in real-time on live, motile cells. And I think their microscope comes close to being able to do this.”
Janetopoulos’ work is yet unpublished, but a paper in the March 22 issue of Science featured work by a research group at Stanford University led by Roel Nusse that utilized the Bessel beam technique. The group examined how a protein called a Wnt is believed to maintain the self-renewal of embryonic stem cells. There is an entire family of Wnt proteins that play an important role in embryonic development, stem cell maintenance, tissue regeneration, bone growth, stem cell differentiation and many human cancers. When embryonic stem cells divide, their daughter cells may become non-embryonic stem cells that perform more specific functions. But in the presence of specific Wnt proteins, there appear to be more embryonic stem cells among the daughters.
3D CELL DIVISION: The stages of cell division, seen frequently in 2-dimensions, imaged with the Bessel beam plane illumination: chromosomes appear in orange and microtubules in green. The Bessel beam technique offers high spatial resolution in the Z-axis, providing detailed 3D movies. Credit: Liang Gao and Eric Betzig, Janelia Farm
The Stanford group wanted to see exactly how this relationship played out. Shukry Habib, a postdoctoral researcher in Nusse’s group, developed a microscale bead that can attach to a Wnt (specifically the Wnt3a protein) and track its motion and orientation. With Betzig’s technique he observed the cells dividing, and could measure precisely the orientation of the cell in the presence of the Wnt protein. With the Bessel beam, the researchers observed how the Wnt influenced structural components of the cell that determined where the axis of division appeared. Habib says this is the best resolution footage he’s seen of embryonic stem cell division. He adds that when he presents the movies at meetings, “I can hear people [in the audience] say, ‘wow’—they’re fascinated,” and they want to know how to get in touch with Betzig.
The scientists who have used the Bessel beam technique still have wish lists: such as even faster frame rates, better 3-D and thicker samples. Another popular technique for 3D cell imaging called spinning disc confocal microscopy can’t image samples nearly as long as Betzig’s technique, but it is still better for imaging thick cells and tissue. And for the moment it is also more widely available. Betzig says Bessel beam plane illumination microscopy is “ready” for commercialization. Now he’ll have to wait for a company that wants to manufacture it. Betzig feels he’s hit a sweet spot with his new device; only time will tell just how sweet it is.