Cell News 4/2014
5
The development of fluorescence imaging techniques that bypass the resolution limit of light microscopy had great impact on cell
biological research during the last decade. The community profits tremendously from the increasing availability of robust “super-
resolution” techniques, the application of which can result in a new quality of data and understanding of cell biological processes.
As defined by Ernst Abbe in 1873, the resolution limit in light microscopy is generally limited to approximately half the wavelength
of visible light. Simply speaking, the fatness of the pen (“point spread function”), which we use to paint the light microscopy image,
obstructs fine drawing of details smaller then the pen-stroke. In fluorescence microscopy a specific signal pattern of a single emission
wavelength generates the image. Regular fluorescence microscopy applies a uniform excitation flow to the sample and uses a single
objective for excitation and emission. Starting already in the 70s, physicists at the University of Heidelberg started to work on the idea
of overcoming the resolution limit in fluorescence microscopy by modulating the homogenous excitation flow coming from a single
objective. A theory was developed by Christoph and Thomas Cremer, which predicted an image of theoretically unlimited resolution in
a set-up, which would enable sample excitation from all angels. It took, however, until 1994 to turn the concept into an idea that was
really working in increasing the optical resolution. In his so-called "4Pi" setup Stefan Hell used a confocal microscopy set-up with to
objectives arranged in a 180°C angle, which excited and detected the sample from opposite sites using interfering lenses. The opposing
signals would show negative interference on the edges of a detected signal but positively interfere with it in its maximum. This resulted
in a steeper but more narrow point spread function. Thus, he could “focus” emitted signals to a volume almost 10 times smaller than
with regular confocal microscopy. Since then, a number of technologies were developed, which all aimed to circumvent the resolution
limit of fluorescence microscopy and to optimise light yield, velocity of detection and sample preservation. Many technologies are now
available to the community of experts and non-experts users. Commercial systems, although still representing a major investment,
cover a number of state of the art super-resolution technologies. Among others, two technologies are most commonly used today,
termed localisation microscopy (including photoactivated localization microscopy, PALM; stochastic optical reconstruction, STORM
and ground state depletion microscopy, GSDIM) and stimulated emission depletion (STED) microscopy. The latter uses a ring-like in-
terfering laser pulse right after the excitation signal to “focus” the airy disk pattern of the excitation light beam to a narrow spot by
stimulated emission depletion of disturbing fluorescence emitted from fluorophores in the reach of diffraction rings outside the spot.
In combination with the 4Pi principle and with suitable fluorophores, todays available STED systems ideally reduce the resolution limit
to the lower nanometer range, reaching close to 100-fold higher resolution compared to regular fluorescence microscopy.
With their work, Stefan Hell and his US colleagues Eric Betzig and William E. Moerner, who developed the PALM principle for super-
resolution microscopy, not only contributed to breaking the barriers of the resolution limit of fluorescence light microscopy. They
also gave an example to the community of cell biologists: don't think about barriers and limits, but focus on ways to circumvent and
overcome these to make new discoveries possible.
* The Nobel Prize in Chemistry 2014
Eric Betzig, Stefan W. Hell, William E. Moerner
"for the development of super-resolved fluorescence microscopy"
Stefan W. Hell - Facts". Nobelprize.org. Nobel Media AB 2014. Web. 14 Nov 2014.
Laudatio
Breaking Barriers – the development of tech-
niques to circumvent the resolution limit of
fluorescence light microscopy
The DGZ congratules
Prof. Dr. Dr. hc. mult. Stefan W. Hell for the
award of the Nobel Price for Chemistry in 2014*
By Oliver Gruss for the board of the German Society for Cell Biology
©Max-Planck-Institut für biophysikalische Chemie
