Overcoming the limits of optical microscopy - the development of super-resolved fluorescence microscopy

When scientists in the 17th century studied living organisms under an optical microscope for the first time, a new world opened up before their eyes. This was the birth of microbiology, and ever since, the optical microscope has been one of the most important tools in the life-sciences toolbox. However, optical microscopy was limited by a physical restriction as to what size structures it was possible to resolve.

In 1873, the microscopist Ernst Abbe published an equation demonstrating how microscope resolution is limited by, among other things, the wavelength of the light. For the greater part of the 20th century, this led scientists to believe that they would never be able to observe things smaller than roughly half the wavelength of light, ie, 0.2 µm. This meant that while scientists could distinguish whole cells and some organelles, they would be unable to resolve things as small as a normal-sized virus or single proteins, or to follow the interaction between individual protein molecules in the cell.

Figure 1: At the end of the 19th century, Ernst Abbe defined the limit for optical microscope resolution to roughly half the wavelength of light, about 0.2 µm.

But Eric Betzig, Stefan W Hell and William E Moerner have found ways to circumvent Abbe’s limit. The equation still holds but, using molecular fluorescence, Betzig, Hell and Moerner independently have overcome the limitation and have taken optical microscopy into a new dimension. Theoretically there is no longer any structure too small to be studied and the optical microscope can now peer into the nanoworld.

How Abbe’s limit was circumvented

Stimulated emission depletion microscopy

Stimulated emission depletion (STED) microscopy was developed by Stefan Hell in 2000. Here, two laser beams are utilised; one stimulates fluorescent molecules to glow, another cancels out all fluorescence except for that in a nanometre-sized volume. Scanning over the sample, nanometre for nanometre, yields an image with a resolution better than Abbe’s stipulated limit.

Hell was convinced that there had to be a way of circumventing Abbe’s diffraction limit, and when he read the words “stimulated emission” in the book on quantum optics, a new line of thought took shape in his mind.

Fluorescence microscopy is a technique where fluorescent molecules are used to image parts of the cell. For instance, they can use fluorescent antibodies that couple specifically to cellular DNA. Scientists excite the antibodies with a brief light pulse, making them glow for a short while. If the antibodies couple to DNA they will radiate from the centre of the cell, where DNA is packed inside the cell nucleus. In this manner, scientists can see where a certain molecule is located. But they had only been able to locate clusters of molecules, such as entangled strands of DNA. The resolution was too low to discern individual DNA strings.

When Stefan Hell read about stimulated emission, he realised that it should be possible to devise a kind of nano-flashlight that could sweep along the sample, a nanometre at a time. By using stimulated emission, scientists can quench fluorescent molecules. They direct a laser beam at the molecules that immediately lose their energy and become dark. In 1994, Stefan Hell published an article outlining his ideas. In the proposed method, so-called STED, a light pulse excites all the fluorescent molecules, while another light pulse quenches fluorescence from all molecules except those in a nanometre-sized volume in the middle (Figure 2). Only this volume is then registered. By sweeping along the sample and continuously measuring light levels, it is possible to get a comprehensive image. The smaller the volume allowed to fluoresce at a single moment, the higher the resolution of the final image. Hence, there is, in principle, no longer any limit to the resolution of optical microscopes.

Figure 2: The principle of STED microscopy.

Developing the first nano-flashlight in Germany, Stefan Hell’s theoretical article did not create any immediate commotion, but was interesting enough for Stefan Hell to be offered a position at the Max Planck Institute for Biophysical Chemistry in Göttingen. In the following years he brought his ideas to fruition; he developed a STED microscope. In 2000 he was able to demonstrate that his ideas actually work in practice, by, among other things, imaging an E. coli bacterium at a resolution never before achieved in an optical microscope.

Figure 3: One of the first images taken by Stefan Hell using a STED microscope. To the left, an E. coli bacterium imaged using conventional microscopy; to the right, the same bacterium imaged using STED. The resolution of the STED image is three times better. Image from Proc. Natl. Acad. Sci. USA 97: 8206-8210.

Detecting a single fluorescent molecule

In most chemical methods, for instance measuring absorption and fluorescence, scientists study millions of molecules simultaneously. The results of such experiments represent a kind of typical, average molecule. Scientists have had to accept this since nothing else has been possible, but for a long time they dreamt of measuring single molecules, because the richer and more detailed the knowledge, the greater the possibility to understand, for instance, how diseases develop.

Therefore, in 1989, when W E Moerner was able to measure the light absorption of a single molecule, it was a pivotal achievement. At the time he was working at the IBM research centre in San Jose, California.

Eight years later Moerner took the next step towards single-molecule microscopy, building on the previously Nobel Prize-awarded discovery of the green fluorescent protein (GFP).

Moerner discovered that the fluorescence of one variant of GFP could be turned on and off at will. When he excited the protein with light of wavelength 488 nanometres (nm), the protein began to fluoresce, but after a while it faded. Regardless of the amount of light he then directed at the protein, the fluorescence was dead. It turned out, however, that light of wavelength 405 nm could bring the protein back to life again. When the protein was reactivated, it once again fluoresced at 488 nm.

Moerner dispersed these excitable proteins in a gel, so that the distance between each individual protein was greater than Abbe’s diffraction limit of 0.2 µm. Since they were sparsely scattered, a regular optical microscope could discern the glow from individual molecules - they were like tiny lamps with switches. The results were published in the scientific journal Nature in 1997.

By this discovery Moerner demonstrated that it is possible to optically control fluorescence of single molecules. This solved a problem that Eric Betzig had formulated two years earlier.

Just like Stefan Hell, Eric Betzig was obsessed with the idea of bypassing Abbe’s diffraction limit. In the beginning of the 1990s he was working on a new kind of optical microscopy called near-field microscopy at the Bell Laboratories. In near-field microscopy the light ray is emitted from an extremely thin tip placed only a few nanometres from the sample. This kind of microscopy can also circumvent Abbe’s diffraction limit, although the method has major weaknesses - the light emitted has such a short range that it is difficult to visualise structures below the cell surface. In 1995 Eric Betzig concluded that near-field microscopy could not be improved much further but continued to ponder whether it would be possible to circumvent the diffraction limit by using molecules with different properties, molecules that fluoresced with different colours.

Inspired by W E Moerner, among others, Eric Betzig had already detected fluorescence in single molecules using near-field microscopy. He began to ponder whether a regular microscope could yield the same high resolution if different molecules glowed with different colours, such as red, yellow and green. The idea was to have the microscope register one image per colour. If all molecules of one colour were dispersed and never closer to each other than the 0.2 µm stipulated by Abbe’s diffraction limit, their position could be determined very precisely. Next, when these images were superimposed, the complete image would get a resolution far better than Abbe’s diffraction limit, and red, yellow and green molecules would be distinguishable even if their distance was just a few nanometres. In this manner Abbe’s diffraction limit could be circumvented. However, there were some practical problems, for instance a lack of molecules with a sufficient amount of distinguishable optical properties.

A breakthrough came in 2005, when Betzig stumbled across fluorescent proteins that could be activated at will, similar to those that Moerner had detected in 1997 at the level of a single molecule. Betzig realised that such a protein was the tool required to implement his idea - the fluorescent molecules did not have to be of different colours, they could just as well fluoresce at different times.

Figure 4: The principle of single-molecule microscopy.

Just one year later, Betzig demonstrated, in collaboration with scientists working on excitable fluorescent proteins, that his idea held up in practice. Among other things, the scientists coupled the glowing protein to the membrane enveloping the lysosome. Using a light pulse the proteins were activated for fluorescence, but since the pulse was so weak only a fraction of them started to glow. Due to their small number, almost all of them were positioned at a distance from each other greater than Abbe’s 0.2 µm diffraction limit. Hence the position of each glowing protein could be registered very precisely in the microscope. When their fluorescence died out, a new subgroup of proteins could be activated. Again, the pulse was so weak that only a fraction of the proteins began to glow, whereupon another image was registered. This procedure was then repeated over and over again.

Figure 5: The centre image shows lysosome membranes and is one of the first ones taken by Betzig using single-molecule microscopy. To the left, the same image taken using conventional microscopy. To the right, the image of the membranes has been enlarged. Note the scale division of 0.2 micrometres, equivalent to Abbe’s diffraction limit. The resolution is many times improved. Image from Science 313:1642-1645.

When Betzig superimposed the images, he ended up with a super-resolution image of the lysosome membrane. Its resolution was far better than Abbe’s diffraction limit.

The methods developed by Eric Betzig, Stefan Hell and W E Moerner have led to several nanoscopy techniques and are currently used all over the world and the development of super-resolved fluorescence microscopy has won the three researchers this year’s Nobel Prize in Chemistry