Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization

Abstract

Expansion microscopy (ExM) is a novel method that allows super-resolution imaging with conventional microscopes(1, 2). It consists in soaking the cells with a polymer, inducing the polymerization to form a dense meshwork throughout the cell, cross-linking the fluorophores to the polymer and, after digestion of cellular protein, rehydrating of the sample. The swelling of the polymer gel led to a fourfold isotropic stretching of the sample. Therefore, it increases the distance between two objects that otherwise couldnot be seen as two different things with an ordinary microscope. One of the drawback of such a technique is the long preparation made of several stages, i.e. immunostaining, gelation, digestion and expansion. They are really crucial steps for a good imaging post-expansion.In our work we present a comparison between ExM and stimulated emission depletion (STED) nanoscopy(3). Our aim is to study the e possible combination of STED and ExM as a method to further enhance the final resolution achievable. We will in particularly take advantage of the use of separation of photons by lifetime tuning (SPLIT) STED (4).We show application of these methods from single fixed cells to slices of fixed mouse retina tissue.We are also interested in applying the approach to high-density compartments like the cell nucleus to decipher the high-order structure organization of chromatin-DNA.1. Chen, F., P.W. Tillberg, and E.S. Boyden. 2015. Optical imaging. Expansion microscopy. Science. 347: 543-548.2. Chozinski, T.J., A.R. Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L. Wong, and J.C. Vaughan. 2016. Expansion microscopy with conventional antibodies and fluorescent proteins. Nat Meth. 13: 485-488.3. Bianchini, P., C. Peres, M. Oneto, S. Galiani, G. Vicidomini, and A. Diaspro. 2015. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360: 143-150.4. Lanzano, L., I. Coto Hernandez, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini. 2015. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nature Communications. 6: 6701.