Using an exquisitely thin sheet of light, a newly developed microscope reveals the 3-D shapes of cellular landmarks in unprecedented detail. The technique images live cells at high speed so that researchers can create dazzling movies that make biological processes, such as cell division, come alive. Dubbed Bessel beam plane illumination microscopy, the technique was invented by scientists at Howard Hughes Medical Institute’s Janelia Farm Research Campus and is described in an article published online March 4 in Nature Methods. Liang Gao, Thomas Planchon and Eric Betzig display their new Bessel beam plane illumination microscope at Howard Hughes Medical Institute’s Janelia Farm Research Campus. (Photo: HHMI) A major goal of biologists is to understand the rules that control molecular processes inside a cell. If one is trying to learn the rules of a game, it is better to have a movie of people playing the game than it is to have still photos – and the same is true for cells, said Janelia Farm group leader Eric Betzig. Despite advances in microscopy during the past few decades, the field is still hindered by the fact that many techniques require that cells be killed and fixed in position for imaging, he added. There is only so much one can learn from studying dead cells – the equivalent of still photos. Microtubules (green) surround nuclei (red) in a pair of live human osteosarcoma cells. Planes at top show location of cutaway views at bottom. Scale Bars = 5 μm. (Photo: Eric Betzig, Thomas Planchon and Liang Gao) Betzig wanted to create a microscope that would enable researchers to see the dynamic inner lives of living cells. The notion of studying live cells, stippled with fluorescently labeled proteins and other molecules, is not new. But live-cell techniques can be problematic because light produced by microscopes can damage the cell over time. Besides cell damage, light causes the fluorescent molecules – of which there are only so many – to wink out over time. In other words, the longer you study the cell to uncover its properties, the more damage you do to it and the more likely you are to spend your “photon budget,” Betzig said. Moreover, the light of a microscope exposes more of the sample than just the small portion that is in focus. Illuminating the out-of-focus regions produces blur, making small intracellular features appear as lengthened blobs rather than as sharp dots. “The question was, is there a way of minimizing the amount of damage you’re doing so that you can then study cells in a physiological manner while also studying them at high spatial and temporal resolution for a long time,” Betzig said. Dynamics of actin-based filopodia at the surface of a live HeLa cell, at 6-s intervals, show filaments that wave (magenta and yellow arrowheads), extend outward (cyan arrowhead) or retract inward (green arrowhead). Scale Bar = 5 μm. (Photo: Eric Betzig, Thomas Planchon and Liang Gao) Long before arriving at Janelia Farm in 2006, Betzig began thinking about ways to improve live-cell microscopy. He put those thoughts on hold while he focused on designing new microscopy techniques that would ultimately shatter the limits of spatial resolution. Until recently, microscopes could see objects no smaller than 200 nm in size. Several years ago, Betzig and a colleague, Harald Hess, invented photoactivated localization microscopy (PALM), a so-called “superresolution” technique that can produce images of objects as small as 10 to 20 nm. PALM and most other microscopes work by exposing the sample through one objective lens and then collecting the light that comes back through that same lens. That approach causes light to damage the sample and induces blur, making it difficult to observe live cells. In 2008, Betzig began working on ways to overcome these challenges. One idea he had was to use plane illumination microscopy. First proposed about 100 years ago, plane illumination involves shining a sheet of light through the side of the sample rather than on the top. To do that, microscopists use two different objective lenses that are perpendicular to one another. “Because you come from the side, plane illumination confines the excitation much closer to the part that’s in focus,” Betzig said. Although other researchers have used plane illumination to great effect to study multicellular organisms hundreds of microns in size, the light sheets were still too thick to work effectively for imaging within single cells only tens of microns in size. The main problem is that the wide swath of light used in plane illumination exposed more of the cell than Betzig’s group wanted. This caused excessive blur and light toxicity. To circumvent this problem, his group used a Bessel beam, a special type of nondiffracting light beam studied by physicists in the late 1980s and used today in applications including bar-code scanners in supermarkets. Sweeping the beam across the sample creates a thinner light sheet. Montage of live cells imaged by Bessel beam plane illumination microscopy. Clockwise from upper left: internal architecture and vacuoles in a monkey kidney cell (gold); membrane ruffles at the surface of a monkey kidney cell (orange); microtubules in a pig kidney cell (green); mitochondria in a human osteosarcoma cell (blue); and chromosomes during mitosis of a pig kidney cell (orange). (Photo: Eric Betzig, Thomas Planchon and Liang Gao) Bessel beams behave a bit strangely, however, and this is what has kept Betzig’s postdoctoral researchers — Thomas Planchon and Liang Gao — busy over the past few years. Although they produce a very narrow light beam, Bessel beams also create somewhat weaker light that flanks the focal point, making the pattern of illumination look like a bull’s-eye. The extra light lobes are a hindrance because they excite too much of the sample. To compensate for this, Betzig’s group used two tricks. The first is a concept called structured illumination, where, instead of sweeping the beam continuously, they turned it on and off rapidly. This creates a periodic grating of excitation that can be used to eliminate any out-of-focus blur. The other strategy was two-photon microscopy, a method commonly used in neuroscience to visualize thick pieces of brain tissue. One advantage of two-photon microscopes is that very little fluorescence signal is generated from weakly exposed regions. Thus, when the group applied two-photon methods, the background from the Bessel side lobes was eliminated, and all that remained was the light from the narrow central part of the Bessel beam. The ruffled membrane of a monkey kidney cell, as observed by Bessel beam plane illumination microscopy. (Photo: Eric Betzig, Thomas Planchon and Liang Gao) The researchers then set out to image as fast as possible. The Bessel beam swept quickly through the sample, allowing the group to take nearly 200 images per second and build 3-D stacks from hundreds of 2-D images in 1 to 10 s. As they had hoped, they found that they could take hundreds of 3-D image sets without harming the cell, generating amazing movies of cellular processes such as mitosis, where chromosomes divide as one cell becomes two. “There’s no other technique that comes close to imaging as long with such high spatial and temporal detail,” Betzig said. Last summer, as soon as they got their first live-cell images, Betzig, Planchon and Gao took the new instrument to the Woods Hole Marine Biological Laboratory in Massachusetts for a physiology course, where they worked with co-authors Jim and Cathy Galbraith from the National Institutes of Health. “We learned a lot about what works and what doesn’t and ways to treat the cells in a way that maintains their physiological state while we'’e doing the imaging,” Betzig said. “Like every microscope, the instrumentation is only part of the puzzle. A lot of it is finding the right samples and right preparation methods to make it work.” The new microscope also is exciting because it may be used in the future to improve superresolution microscopy. PALM and other superresolution techniques are limited to looking at thin, dead samples and can be very damaging when looking at live ones. “That’s what’s really great about the Bessel – we can confine that excitation and really start to think about applying superresolution microscopy to study structure or dynamics in thicker cells,” Betzig said. Even without superresolution, Bessel beam plane illumination microscopy will be a powerful tool for cell biologists, he added, because it noninvasively images the rapidly evolving 3-D complexity of cells. For more information, visit: www.hhmi.org