Scientists at Howard Hughes Medical Institute’s Janelia Farm Research Campus have developed a 3-D super-resolution microscope to capture intracellular biological processes in real time. The system incorporates a thin, nondiffracting beam of light—called a Bessel beam—into a century-old imaging technique called plane illumination.
Eric Betzig and his research team at Janelia Farm developed the Bessel beam plane-illumination microscope and has already taken detailed 3-D movies of dividing chromosomes, the ruffling movement of the outer surface of a cell, and the dynamic extending and retracting action of filopodia on the surface of a HeLa cell.
To develop the microscopy technique, Betzig’s group started with plane-illumination microscopy, which uses two lens to illuminate a specimen through the side while collecting that light from above. This confines light exposure to the focal plane, reducing out-of-focus areas collected by the detection lens, eliminating phototoxicity and photobleaching outside of the focal plane, and providing a more detailed image from the focal plane. While effective for imaging multicellular organisms hundreds of micrometers in size, the focal plane is dictated by the thickness of the light beam, known as a Gaussian beam.
“There’s a tradeoff between how thick the beam is and how long or flat the sheet of light is before it starts to diffract and cause blurring of the image and phototoxicity within the cell,” said Betzig. “If a cell is 50 microns in diameter, the thinnest light sheet you can attain using a Gaussian beam would be about 3 microns thick, and that’s still fairly large on a cellular scale.”
In contrast, a Bessel beam never diffracts, since its thickness is independent of the length and thinness of the light sheet. By sweeping this beam across a specimen, Betzig created thin sheets of light that could be compiled to create a 3-D image.
Although Bessel beams produce a very long narrow light beam, they also create weaker areas of light that surround the focal point. “There’s a bright central peak, and then there’s concentric circles of light around it that contain significant energy as well,” said Betzig. These weaker areas of light form side lobes that sweep through the specimen and excite areas outside of the focal plane.
To overcome this problem, Betzig devised two methods: structured illumination (SI) and two-photon microscopy (TPM). SI creates a periodic grating pattern of excitation by turning the Bessel beam on and off as it sweeps through the specimen, which reduces the extra light of the side lobes and increases the resolution of the image. Weakly exposed regions of the specimen generate very little fluorescent signal using TPM, a fluorescent imaging technique for deep living tissue, eliminating the background fluorescence generated by the Bessel side lobes.
Currently, Betzig is collaborating with other cell imaging groups in order to showcase the technique’s myriad applications. One collaboration seeks to elucidate the functions and interactions of various proteins involved in the mitotic process. Another will examine the cellular processes that govern stem cell migration and differentiation in 3-D matrices. “That sort of motility also drives metastasis of cancer from one site to another. It’s a hot area, and this will be a great tool for studying that,” says Betzig.
The paper “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination” was published 4 March 2011 in Nature Methods.