Scientists at Howard Hughes Medical Institute’s Janelia Farm Research Campus have developed a new microscopy technique that uses light to virtually slice through living cell samples. Known as Bessel beam plane illumination microscopy, the technique currently produces slices less than 0.5 μm at a rate of over 200 per second. The high rate of slicing allows for movies to be made of the inner workings of living cells. See for yourself:
Although other researchers, including Janelia Farm Fellow Philipp Keller, 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 non-diffracting 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, his group found.
Bessel beams behave a bit strangely, though, 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 problem, 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, like firing a machine gun. This creates a periodic grating of excitation that can be used to eliminate any out-of-focus blur. (Structured illumination, used by Janelia Farm Group Leader Mats Gustafsson, is also one way of achieving super-resolution.)
Another strategy Betzig’s group used is two-photon microscopy, a method commonly used in neuroscience to visualize thick pieces of brain tissue. One of the advantages of two-photon microscopes is that very little fluorescence signal is generated from weakly exposed regions. Thus, when they 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.
Full story with more videos: New Microscope Produces Dazzling 3D Movies of Live Cells…
Abstract in Nature Methods: Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination
(hat tip: PopSci)