My focus of studies at KTH is biomedical physics. It is mainly about how one can use the laws of physics in order to gain a better understanding of biological processes in organisms. These processes are so fascinating, because they take place on very tiny scales while affecting the body as a whole. The problem with that is, that they are so tiny, that we cannot observe them with our eyes, neither can we with ordinary light microscopes. And here comes super-resolution optical microscopy into place, which I want to try to explain to you in this article.

The reason that I write about this topic is, that Stefan Hell, who received the 2014 Nobel prize in chemistry for his research on super-resolution optical microscopy, recently gave a talk at KTH. One year after being awarded the Nobel prize, Hell returned to Stockholm, because he becomes honorary doctor at Kungliga Tekniska Högskolan.

How Does a Super-Resolution Microscope Work?

The basic procedure of most high-resolution microscopy is to focus a beam on a spot of the sample. The beam can consist of light, electrons or any other particles and one observes the interaction of the beam with the sample – in simple words: if some light comes back, we hit something, if not, then not. With this spot we can scan the whole sample and thereby reconstruct a picture of it. It is clear, that the smaller the spot, the clearer will be the image.

The problem with conventional light microscopes is, that due to diffraction, the size of the spot cannot be smaller than half of the wavelength used. So when using a laser beam with a wavelength of 630 nm, two points in the sample that are closer than 315 nm cannot be resolved. Nanometers are already very tiny, but why not go further?

Beyond the Limit

For more than a hundred years this has been thought to be impossible and nobody even tried to break the Abbe diffraction limit. But many things have fundamentally changed in the world of physics since the establishment of  quantum mechanics in the beginning of the last century. So Stefan Hell believed that there must be a way – and he found one, a very clever one to be precise.

(left) Exciting laser beam, (middle) Depleting laser beam, (right) Resulting Excitation | Image by Marcel Lauterbach under CC-BY-SA 3.0 license

STED Microscopy: (left) Exciting laser beam, (middle) Depleting laser beam, (right) Resulting Excitation if both beams overlap | Image by Marcel Lauterbach under CC-BY-SA 3.0 license

His idea was to accept the fact, that a light spot was diffraction limited, but to use a second, doughnut shaped light beam to black out the boarders of the first spot in order to make it more and more tiny. To be honest, this sounds like cheating on the laws of physics. But thus it is super fascinating to find out, why it is not. To understand this, we have to get a little deeper in how light is emitted after the laser beam has hit the molecule.

Let There Be Light

Around the nucleus of a molecule, electrons are moving in discrete shells. The wider the orbit, the more energy one electron has. When one shines laser light on the molecule, energy is deposited and the electrons are lifted to higher shells. After some time the electron spontaneously hops back to its original shell and thereby itself emits light. This so called fluorescence light will be detected by the microscope.

Actin Filament of a Cell shown under STED microscope | Image by Howard Vindin under CC BY-SA 4.0

The inside of a cell: actin filament shown under a STED microscope | Image by Howard Vindin under CC BY-SA 4.0

But how can we use a second, doughnut-shaped light beam in order to inhibit the emission of fluorescence light? If the second beam hits an electron that is already excited, it is kicked down immediately under the so called stimulated emission. In contrast to the fluorescence light, the stimulated emission has the same wavelength as the second light beam and can therefore easily be filtered out. What remains is a very tiny spot of fluorescence light in the middle.

Thus the second light beam works just like a vacuum cleaner that continuously switches all molecules off except for those in the middle of the doughnut. And because the width of the middle of the doughnut is not diffraction limited, resolutions of up to 2.4 nm (instead of 315 nm) have been reached.

This kind of microscopy is called stimulated emission depletion microscopy (STED) and has been proposed by Stefan Hell in 1994 and experimentally realized in 1999.

More To See

The talk has been recorded on video and can be viewed from the website of the Albanova University center. Make sure to also have a look at other talks published there. If something remains unclear or you have some further notes, feel free to leave a comment below! You can also head over to my Facebook page.