Expanding our view with Expansion Microscopy
The resolution of lens-based optical microscopy is limited by light diffraction and does not allow resolving objects closer to each other than approximately 200 nanometres, according to Abbe’s equation. The diffraction barrier was surpassed by the development of super-resolution microscopes, which are based on switching fluorescent molecules on and off sequentially, and recording the signal from one point at a time. This approach allows distinguishing features closer than the diffraction limit, and can reach resolutions of tens of nanometers or less. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, William E. Moerner and Stefan W. Hell for “the development of super-resolved fluorescence microscopy.”
Now, researchers from the Massachusetts Institute of Technology have taken a new approach to microscopy: instead of aiming to optically magnify the images of structures, they do a physical magnification of the specimen itself. This novel method, termed expansion microscopy, involves synthesizing a polymer network within the biological specimen and expanding this mesh by introducing water into the system. The sample can then be visualized at super-resolution capabilities with conventional microscopes.
First, antibodies coupled to fluorescent molecules are used to label the cellular components of interest. Then, the polymerization reaction is initiated and the polymer, with the fluorescent labels integrated, becomes embedded into the tissue. In the next step, the tissue is digested, as it would hinder the expansion, leaving behind the polymer network with the fluorophores that mark the localization of proteins. The addition of water expands the sample about five times, evenly in all three dimensions, and this preserves its native structure and the spatial arrangement of its components. Therefore, two proteins located closer than the diffraction limit can be optically resolved with conventional light microscopes, by employing fluorescent labels integrated into a swellable polymer network. As a proof of concept, microtubules and clathrin-coated pits in cultured cells and synapses in the brain tissue were visualized at a resolution of 60 nm, without affecting the physiological structures, as evidenced by the pre and post-expansion images.
Chen F, Tillberg PW, Boyden ES. Science (2015). Vol. 347 no. 6221 pp. 543-548 DOI: 10.1126/science.1260088
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