Multicolor 3D Images of Proteins

Super-resolution micro­scopy is a technique that can see beyond the diffrac­tion limit of light. The technique has garnered increasing interest recently, especially since its developers won the Nobel Prize in Chemistry in 2014. By exploiting fluores­cence, super-reso­lution micro­scopy now allows scientists to observe cells and their interior structures and orga­nelles in a way never before possible.

Human centrioles labelled with antibodies against two proteins and imaged using super-resolution microscopy. From many individual particles showing projections of the centriole complex in various orientations, by using a fused intermediate, the newly developed method allows now to reconstruct a multicolor 3D model. (Source: C. Sieben, EPFL)

Many of the molecular complexes inside cells are made up of multiple proteins. Since current techniques of super-resolution micro­scopy typically can only use one or two fluores­cent colors, it is difficult to observe different proteins and decipher the complex archi­tecture and underlying assembly mechanisms of the cell’s interior structures. An even greater challenge is to overcome the noise inherent to the super-reso­lution methods and fluores­cent labeling, to achieve the full reso­lution potential.

Scientists from the lab of Suliana Manley at EPFL have now solved both problems by developing a new method to analyze and recon­struct super-reso­lution images and re-align them in a way that multiple proteins can be placed within a single 3D volume. The method works with images taken with large field-of-view super-reso­lution micro­scopy, with each image containing hundreds of two-dimen­sional projec­tions of a labeled structure in parallel.

Each 2D view represents a slightly different orien­tation of the structure, so that with a dataset of thousands of views, the method can computa­tionally reconstruct and align the 2D images into a 3D volume. By combining infor­mation from a large number of single images, the noise is reduced and the effective reso­lution of the 3D recon­struction is enhanced.

With the help of Pierre Gönczy’s lab at EPFL, the researchers tested the method on human centriole complexes. Centrioles are pairs of cylin­drical molecular assemblies that are crucial in helping the cell divide. Using the new multi­color super-resolution recon­struction method, the researchers were able to uncover the 3D archi­tecture of four proteins critical for centriolar assembly during organelle bioge­nesis.

The new approach allows for unlimited multi­plexing capa­bilities. “With this method, if the proteins in the structure can be labeled, there is no limit to the number of colors in the 3D recon­struction,” says Manley. “Plus, the recon­struction is inde­pendent of the super-reso­lution method used, so we expect this analysis method and software to be of broad interest.” (Source: EPFL)

Reference: C. Sieben et al.: Multicolor single-particle reconstruction of protein complexes, Nat. Meth. 15, 777 (2018); DOI: 10.1038/s41592-018-0140-x

Link: Laboratory for Experimental Biophysics, Institute of Physics, École Polytechnique Fédérale de Lausanne EPFL, Lausanne, Switzerland

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