Scientists develop formula to investigate 'nanocosmos' of cells
Scientists from the Max Planck Institute (MPI) have developed a new formula to reduce the resolution of light microscopes to 15 nanometres. It beats a previous formula, which held that optical resolution is impossible below 200 nanometres. Scientists say that the new formula could reveal how the 'nanocosmos' of cells works. Since its discovery in the 17th century, the light microscope has been the key to new biological and medical discoveries. Light, however, propagating as a wave, is subject to the phenomenon of diffraction, which limits the resolution of the object under the microscope. These resolution-limiting effects were first described by in 1873 by Ernst Abbe, a German physicist, who observed that structures which were closer to each other than 200nm could not be visually separated when observed using visible light; when viewed through the optical microscope they are perceived as a blurred, single entity. The physicist's recognition of the limited resolution of light microscopes was long thought to be an unalterable law of far-field light imaging. Achieving higher resolution required the use of an electron microscope. That all changed several years ago when researchers in the department of NanoBiophotonics at the MPI for Biophysical Chemistry in Göttingen developed a technique known as Stimulated Emission Depletion (STED) microscopy, which, they claimed, was able to break the Abbe resolution limit. It involves two overlapping laser beams. To make the fluorescent spot smaller, researchers use a light beam to excite the florescence dyes that are attached to a protein sample. Before the excited molecules in the light can fluoresce, a smaller second beam is used which overlaps the first and forces the molecules in the outer ring-beam to relax. In other words, those molecules in the clearly smaller spot in the centre of the light ring remain excited and fluoresce. In April, a Göttingen-based research team verified this new law with experiments to visualise the protein synaptotagmin, which is embedded in the membranes of individual vesicles in cells. Vesicles are membrane 'bubbles' roughly 40 nm in diameter filled with neurotransmitters, which transport chemical messenger molecules to synapses, the contact points between nerve cells, enabling nerve signals to pass between cells. Their contents are released at the synapse when the vesicle membranes fuse with the membrane of the nerve cell. Previously it was unclear whether the proteins sticking in the vesicle membrane spread out over the cell membrane after the fusion event, or whether they remained together, localised in the membrane patch, which previously formed the vesicle. With the aid of STED microscopy, the researchers were able to show that the synaptotagmin molecules of a single vesicle remain together after fusion. The latest experiments, the results of which were published in the proceedings of the National Academy of Sciences, now show that the STED technique can reach a resolution as low as 15nm. The ability to view cells at such a nanoscale is expected to increase understanding of the intricate workings of cells. For example, the formula will shed light on the functioning of proteins, which with their dimensions of 2-20 nanometres were previously too small to view. But 'the full potential of the STED technique has still not been fully exploited', according to Professor Stefan Hell. He claims that the resolution of the size order of a dye molecule is imaginable - this corresponds to a sharpness of one or two nanometres.
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