Looking at the inside of cells with flashes of light

How glowing proteins help to overcome the diffraction limit

Photo: Image of Cytoskeleton with PALM (Courtesy of Zeiss and sowie S. Niwa, N. Hirokawa, University of Tokyo, Japan)

How do synapses grow between neurons? What part do proteins play in the evolution of life-threatening diseases? All of these questions can be addressed with super-resolution microscopy. You can picture structures of only 20 nanometers in size, far beyond the diffraction-limit, described by Ernst Abbe in the 19th century. For the development of that technology the Nobel Prize in chemistry 2014 was awarded to Eric Betzig, Stefan Hell and William Moerner.

The method developed by Betzig and his colleague Harald Hess – Photoactivated Localization Microscopy (PALM) – is particularly suitable for the examination of living cells. The ZEISS microscope ELYRA works with this method. In PALM, very short flashes of light stimulate fluorescent proteins to glow. The short duration of the flashes means that only a few proteins begin to glow. Pictures are taken until the intensity of the fluorescence has dropped to low levels. After that, a new light-flash stimulates other proteins.

As it is not possible to separate light sources that are too closely packed according to the diffraction limit of Abbe it is necessary to stimulate only a few proteins for each picture. More widely separated proteins can be located even if they appear blurred by diffraction. With mathematical algorithms, these effects can be removed.

With frequent repetition of the procedure, every protein glows once. Adding up the individual protein positions yields an image of the complete cell. Therefore, the diffraction limit of the single frames can be overcome and processes inside cells will no longer be a secret.

More information about ZEISS
More information about the topic: ZEISS press release in response to the Nobel Prize 2014