Resolution of Fluorescing Microscope Reaches 15nm

Scientists have used a new trick involving fluorescent dyes to surpass their own STED (stimulated emission depletion) microscope resolution records set in April and further distance themselves from limits imposed by Abbe's Law.

Scientists at the Max Planck Institute for Biophysical Chemistry in Göttingen said they have further opened the door to the nanocosmos of the cell by improving the resolution of STED microscopes to 15 nm, or 12 times sharper than a conventional microscope. In April, the same team of scientists, led by Professor Stefan Hell, achieved an in-cell detail sharpness of 60 nm. The intensive light required for that level of sharpness could only be achieved because a new trick prevents the fluorescent dyes from extensive bleaching, allowing the researchers to further decrease the microscope's effective focal spot.

Figure 1: A look at the inside of cells becomes sharper: Both figures show the filaments in a human nerve cell; with a common confocal microscope (left), and with a STED microscope plus mathematical deconvolution. The resolution of the STED microscope is better by more than an order of magnitude. (Images: Max Planck Institute for Biophysical Chemistry)

Fluorescence microscopy is most often used in biology. Its advantage lies in the fact that the inside of living cells can be observed without destroying the cell. With a fluorescence microscope, fluorescent markers are attached to proteins and other biomolecules so that scientists can observe the marker. For a long time, low resolution prevented a deeper look into how viruses infect a cell, how nerve cells transport signals or how proteins work -- single proteins with their dimension of 2-20 nm diameters were, until now, just too small.

Only a few years ago, physicists believed that it was impossible to resolve details that lie closer together than 200 nm. This limit is imposed by Abbe’s Law, which states that the resolution of a light microscope cannot be more accurate than half of the wavelength of light entering the microscope.

Hell and his co-workers overcame this limit with a trick: They excite the fluorescence dyes that have been attached to the proteins with a blue light beam. The size of the spot from this beam though cannot be made sharper than 200 nm, as governed by Abbe’s Law. However, before the excited molecules in the light spot can fluoresce, the molecules in the outer section of the light spot are forced to relax. To this effect, they overlap a second ring-shaped light beam, the STED beam, over the excitation spot. This now means that only those molecules in the clearly smaller spot in the center of the light ring remain excited and can finally glow.

STED microscopy: The excitation light beam (EXC beam, in blue) is steered by a mirror through the objective lens, and due to diffraction is focused to a spot ca. ~200 nm in diameter on the sample. The excitation light excites fluorescent markers which tag molecules of interest (e.g. proteins) in the sample. The markers are excited to a higher energy state, from which they emit light of a longer wavelength (via fluorescence decay) when they return to the ground state. By scanning this blue excitation spot over the sample (the cell) and recording the emitted fluorescent light with a computer, one can form an image of the sample. The smaller the excitation spot is, the higher the resolution of the microscope. However, due to diffraction, the excitation spot cannot be made smaller than ~200 nm by focusing with a lens. The trick with STED microscopy is that one uses a second beam (STED beam, in orange) to quench the fluorescent markers before they fluoresce. Because the STED beam is doughnut-shaped and centered over the excitation spot, one is able to preferentially quench the markers at the outer edge of the excitation spot and not those in the center. The result is a smaller effective fluorescence spot (green), here reduced to a diameter of ~66 nm. By making the STED doughnut very intense, it is in principle possible to shrink the fluorescent spot to molecular size, thus attaining molecular resolution.

The more intense the STED beam, the smaller is the spot in which the molecules can still fluoresce. The resolution is also improved. However, the fluorescing dye molecules also bleach faster in an intense light beam and one sees...nothing. The scientists discovered that the fluorescing molecules bleach, for the most part, because they are continually transferred for a microsecond into a further dark state, what physicists call a "triplet state." For a molecule in this state, being hit by a light particle makes it irreversibly bleached. The solution to the problem is to irradiate the molecule with a light pulse that allows 4 microseconds between the pulses -- enough time for the molecules to return from the dark state. Subsequently, the molecules are available once again for excitation and relaxation.

"The full potential of the STED technique has still not been fully exploited," said Hell. Resolution of the size order of a dye molecule is imaginable -- this corresponds to a sharpness of 1-2 nm. With their STED technique, the scientists in Göttingen have already shown how the protein Bruchpilot is spatially concentrated in synapses, and how it triggers the building of active synaptic zones at which the nerve cell selectively releases neurotransmitter. They also explored how, during synapse, the released proteins assemble at the presynaptic membrane.

The work of Hell and his colleagues, "Macromolecular-scale resolution in biological fluorescence microscopy", is described in the August issue of Proceedings of the National Academy of Sciences.

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