Expansion Microscopy

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Biomolecules, such as nucleic acids, lipids, and proteins, are nanoscale in size, and often localized with nanoscale precision with respect to each other, and to cellular structures. Analyzing the nanoscale configurations of biomolecules in cells and tissues is critical for understanding how they work, as well as how they go wrong in disease states. Not surprisingly, much effort has been devoted to inventing methods (e.g., super-resolution microscopy, cryo-electron microscopy) for nanoimaging biological specimens, primarily in their preserved state. However, all of these technologies require expensive equipment, and specialized skillsets. Given that all biological systems involve nanoscale building blocks and their interactions, a major question is whether nanoimaging can be democratized, so that anyone could do it, without expensive equipment or extensive training. This grant is a first competitive renewal of our group’s primary grant that supports the development of a technology that we think could potentially meet this goal. We recently announced that in contrast to all previous methods for imaging preserved biological specimens, which magnify their images, specimens could themselves be physically magnified. This technology, which we call expansion microscopy (ExM), involves equipping key biomolecules or labels within a specimen with anchoring molecules, then densely and evenly permeating the biological specimen with a mesh of swellable polymer (that binds to the anchors, thus anchoring key biomolecules or labels to the polymer), softening the specimen to disrupt endogenous molecular interactions, and adding water to swell the polymer, which in turn pulls the biomolecules or labels apart from each other. The process is even down to the nanoscale, and thus enables nanoimaging of cells and tissues on ordinary microscopes. In addition, several recent papers point to an additional advantage of ExM – by pulling biomolecules apart from each other, you decrowd them for better labeling by fluorescent probes, sometimes turning invisible biomolecules into visible ones. ExM is already in use by many hundreds of research groups, with over 250 experimental preprints and papers appearing to date. Here we propose to make ExM simpler, more powerful, faster, more applicable to human samples, and more precise in resolution. Specifically, we will (Aim 1) create a unified, simple, high-speed ExM protocol; (Aim 2) create a unified, simple, high-speed, single-step 20x expansion protocol; (Aim 3) optimize the new unified, simple, and high-speed ExM protocols for human tissues. We propose a fast-paced, 4 year, technology development grant, with the goal of delivering, to the entire biology and medical community, a truly democratized toolbox that enables anyone to do nanoimaging. We will share all protocols as freely as possible both on the web and through protocol papers, as well as through hosting people at hands-on workshops.