![]() ![]() ![]() Like STED, this method exploits the different states of fluorescent probes. Hell shared the Nobel with William Moerner and Eric Betzig, who in 2006 achieved sub-diffraction resolution with a method called single-molecule localization microscopy (SMLM). Soon, superresolution was the leading edge of numerous developments in microscopy and its impact was recognized with a Nobel Prize in 2014. Later, other techniques also worked around Abbe’s diffraction limit with variations on the approach. Hell’s breakthrough technology, which he called stimulated emission depletion (STED), was the first to open the doors into the nanoscopic world. Moving the donut over the sample yielded an image with sub-diffraction resolution.īoom! The mundane donut bumped up the resolution power of microscopes ten-fold and beget the field of superresolution microscopy. Thus, all fluorophores in the donut-shaped area were silenced and the effective area of detectable photon emission was confined to the donut hole. That second beam was at a wavelength that knocked excited fluorophores back down to their ground state. Hell followed excitation with a second beam of light in the shape of a donut. So, to improve resolution some of those fluorophores must be de-excited. All fluorophores within that range become excited, emit photons, and thus, any structures smaller than 200 nm are indistinguishable. He reasoned: the beam of excitation light projects onto the focal plane of a sample in a smear of approximately 200 nm. Focusing on this difference in states was the crux of Hell’s loophole. The fluorescence microscope targets light onto the probes, sending them into a state of excitation, and then detects the photons they emit upon returning to their ground state. Here, molecules of interest in a sample are tagged with tiny fluorescent probes or fluorophores. Hell worked with fluorescence microscopes. Rather than trying to change the optics of a microscope, Stefan Hell exploited a loophole to outsmart the arrogance of light and blast through the resolution barrier. ![]() That is, until someone in 1990s tackled the problem from a different angle. Yet, resolution remained at Abbe’s limit. Fluorescence confocal microscopy did wonders in revealing the inner workings of cells. Of course, microscopes got better over the last century. This immutable fact, called the Abbe diffraction barrier, is literally etched in stone at the Friedrich Schiller University of Jena in Germany, and for decades microscopists sighed in resignation. All molecules within 200 nm from one another appear as one blob under a microscope. That’s 100 times larger than the size of a molecule. In 1873, physicist Ernst Karl Abbe told us that the best we can do is resolve objects that are about 200 nm apart. Zooming in, however, to look at the smallest of things – like molecules bouncing around in cells – is impossible. The diameter of the smear is in the hundred nanometer range. That is, there is an inherent limit to the resolution of a microscope dictated by the very nature of the signal it detects. The result is that when two points of light are close enough to have overlapping smeared blobs, we can’t tell them apart. No matter how sophisticated the instrument, a point of light viewed through a microscope becomes a smeared blob because light waves passing through the aperture of an objective diffract. That latter whim vexes optical microscopy. She refuses to make up her mind about being a particle or a wave. Among the stimuli that evolution chose for us to perceive the world, light is like a self-absorbed celebrity.
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