X10 Expansion Microscopy or: How to do Super-Resolution imaging with diapers

Look at the picture below. What can you see?

You can probably tell that there is a pond, with someone (my humble self) standing in the foreground. You can also probably tell that there are a few trees and a building in the background. But can you tell me how many fingers I’m holding up? How about how many ducks there are, or what is written on the sign I’m holding?

Now look at the picture below.

Something is obviously different. The resolution in this picture is much better than in the first one. But what does this term “resolution” mean?

Simply put, resolution is the minimal distance at which two objects can still be distinguished from each other. When the resolution is on the order of centimeters (like in the first picture) you can’t distinguish any objects that are made up of features less than a few centimeters apart, like the writing on the sign, or the duck to the right of the stones. These objects are below the resolution limit, as we would say in optical physics terms.

Us biologists face precisely this problem when we try to observe cells under a microscope. The only difference is that the resolution we’re interested in is on the order of nanometers, rather than centimeters or millimeters. While a powerful microscope provides a much better resolution than even the most advanced consumer cameras, the physics of light puts a hard limit on the best resolution we can achieve under normal circumstances.

Light behaves like a wave, and it is impossible to focus it precisely onto one arbitrarily small spot. This means that you will always get a light signal from an area rather than a single spot, and your final image will (to a certain extent) be blurred (the degree of the blurring mainly depends on the wavelength of light). This phenomenon is called “Abbe diffraction” and was first described by Ernst Abbe in 1873. His formula to calculate the theoretical resolution of an imaging device is so central to optical physics that it got its own monument in front of the University of Jena (see image below).

The smallest possible limit for the resolution obtainable in light microscopy is accordingly called “the diffraction limit” and is, in practice, approximately 250 nm.

This image is modified from “Ernst-Abbe-Denkmal Jena Fürstengraben”  by Daniel Mietchen

The existence of the diffraction limit means that we typically cannot discern any objects that are closer together than 250 nm using classical light microscopy. 250 nm may sound very small (and it is – 250 nm is about 1/100th the diameter of a human hair!) until you consider that most proteins are only 2.5 nm large. This is, of course, a problem for us biologists.

So then how can we visualize processes and structures within a cell beyond the diffraction limit?

One solution is to move away from photons and turn to electrons. In electron microscopy, we can achieve 1000-fold better resolution than in light microscopy and go down to about 0.25 nm, since the wavelength at which electrons travel is just so much shorter than that of photons in visible light.

The only problem is, electron microscopy images look rather boring and drab (like the synapse shown below) since we can’t get any color in them. What allows us to color a light microscopy image is that we can observe photons traveling at different wavelengths and distinguish them from each other. This isn’t possible in electron microscopy, since all electrons look exactly the same during imaging.

We can take advantage of these differences in the photons to tell apart different proteins in the cell. In order to do this, we mark the proteins with fluorophores that emit different wavelengths of light, and use a fluorescent microscope to separate the fluorophores and color in our images.

This image is reproduced from “A high magnification image of synapse obtained by electron microscopy” by the Okinawa Institute of Science and Technology

This is a synapse, the connection between two nerve cells where they relay signals. The big circle at the top is the sending neuron, the big circle below is the receiving neuron. The little circles in the sending neuron are synaptic vesicles, where neurotransmitter is stored. The darker bar where the sending and the receiving neurons connect is called the active zone (on the sending site) and the post-synaptic density (at the receiving side). This is where neurotransmitters are released or received, respectively.

But now we arrive back at our original problem. How can we improve the resolution in light microscopy so that we can see individual proteins?

Until recently, scientists ‘cheated’ their way around the resolution limit by tricking fluorophores into unlikely molecular states. Switching fluorophores on and off temporally separates their light emissions, allowing us to observe them separately and pinpoint their individual positions. This approach (which is used in STED and STORM microscopy for example) unfortunately requires sizable amounts of two of the most limited commodities for us scientists – time and money. In addition, super-resolution microscopy doesn’t work with all types of fluorophores, which limits the number of color channels we can observe at a high resolution.

In the image below, we can again see a synapse, with three proteins labelled. Only one of them (red) is in super-resolution (about 40-50 nm, in this case), while all the others are at a normal resolution (250-300 nm, in this case).

SVs, synaptic vesicles; AZ, active zone. Adapted from: Wilhelm et al. (2015) Science.

A peculiar solution to these problems arose fairly recently, from a very unlikely direction – baby diapers!

Superabsorbent hydrogels, like the ones found in baby diapers, can swell to many times their original volume when brought into contact with water (see the animation below). If we could expand a biological sample equally in all three dimensions, we would be able to resolve targets that previously were too close together, while still preserving the organization of the sample. The idea is strikingly simple! If cellular features are too close together to resolve, why can’t we pull them apart until we can resolve them?

Image Credit: Wiebke Jahr

In 2015, expansion microscopy was invented when the team of Ed Boyden at MIT found a way to embed biological samples into superabsorbent hydrogels for the first time. They originally achieved 4-fold expansion, which separates features as close together as 70-80 nm far enough to push them beyond the 250-nm limit, and thus resolving them! In the lab of Silvio Rizzoli, I further developed this idea and achieved 10-fold expansion in an approach we termed X10 microscopy.

With X10, we can now resolve targets that originally were only 25 nm apart. Perhaps the best thing about it is that multi-color super-resolution imaging finally becomes easily achievable, almost trivial.

SVs, synaptic vesicles; AZ, active zone; PSD, post-synaptic density. Adapted from: Truckenbrodt et al. (2018) EMBO Rep.

Since we can now do multi-color super-resolution microscopy on conventional microscopes, X10 also saves a lot of time and money. It is particularly important to me that this new approach makes super-resolution microscopy available (and affordable!), not only to a few specialists, but to most biology laboratories.

Various labs are now using X10 to investigate synapses, the cytoskeleton, protein trafficking, organelle organization, receptor signaling, and many more topics in all areas of biology. I hope that X10 will contribute to the ongoing transformation of how we perceive biology through super-resolution imaging.


Abbe E (1873) Die Construction von Mikroskopen auf Grund der Theorie. In: Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für mikroskopische Anatomie.

Chen F, Tillberg PW, Boyden ES (2015) Expansion Microscopy. Science.

Truckenbrodt S, Maidorn M, Crzan D, Wildhagen H, Kabatas S, Rizzoli SO (2018) X10 expansion microscopy enables 25‐nm resolution on conventional microscopes. EMBO Reports.

Truckenbrodt S, Sommer C, Rizzoli SO, Danzl JG (2019) A practical guide to optimization in X10 expansion microscopy. Nature Protocols.

Wilhelm BG, Mandad S, Truckenbrodt S, Kröhnert K, Schäfer C, Rammner B, Koo SJ, Claßen GA, Krauss M, Haucke V, Urlaub H, Rizzoli SO (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science.

Dr. Sven Truckenbrodt

Postdoctoral researcher, Institute of Science and Technology Austria (IST Austria)

Leave a Reply