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Thin sheets of light image live cells

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Compiled by BioPhotonics staff

Using an extremely thin sheet of light, a new type of microscope reveals the 3-D shapes of cellular landmarks in never-before-seen detail. The technique images live cells at high speed, creating movies of biological processes such as cell division.

The ruffled membrane of a monkey kidney cell, as observed by Bessel beam plane illumination microscopy. Images courtesy of Eric Betzig, Thomas Planchon and Liang Gao.

Known as Bessel beam plane illumination microscopy, the method was developed by researchers at Howard Hughes Medical Institute’s Janelia Farm Research Campus. Their findings were published online March 4, 2011, in Nature Methods (doi: 10.1038/nmeth.1586).

Their goal was to design a microscope that would reveal the dynamics of living cells while minimizing the light damage and fading of the fluorescent labels associated with live-cell imaging.

A 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).

Although plane illumination has been used to study multicellular organisms, previously the sheets of light have been too thick to image effectively within single cells only tens of microns in size. The wide swath of light also exposed more of the cell than the scientists desired. To circumvent this, the group used a Bessel beam – a nondiffracting light beam used in applications such as bar-code scanners – to create a thinner light sheet across the sample.

Microtubules (green) surround nuclei (red) in a pair of live human osteosarcoma cells. Planes at top show the location of the cutaway views below. Scale bars = 5 μm.

The team noticed that the Bessel beam behaved a bit strangely, however. Besides a very narrow light beam, the beams also created a weaker light that flanked the focal point, creating an illumination pattern that looked like a bull’s-eye. To counteract this, the team used two tricks: two-photon microscopy and a concept called structured illumination, which turns a beam on and off rapidly over a sample.

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 bars = 5 μm.

To image a sample as fast as possible, they used a Bessel beam to sweep quickly through the sample, taking nearly 200 images per second; almost simultaneously, in 1 to 10 seconds, a three-dimensional stack was built from hundreds of two-dimensional images. The scientists took hundreds of such 3-D image sets without harming the cell, generating movies of mitosis, for example.

The new instrument may be used in the future to apply superresolution microscopy to thicker cells. In any case, this technique’s ability to image the rapidly evolving 3-D complexity of cells noninvasively should make it a powerful tool, the team said.

May 2011
3-D imagingAmericasBessel beamBessel beam plane illumination microscopyBiophotonicsBioScancell biologycell divisionHoward Hughes Medical Institute’s Janelia Farm Research Campusimaginglight beamliving cellsMicroscopymitosismolecular processesmulticellular organismsNewsnoninvasive imagingplane illumination microscopystructured illuminationsuperresolutionsuperresolution microscopytwo-photon microscopyVirginia

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