Expansion microscopy (ExM) is a novel method that allows super-resolutionimaging with conventional microscopes(1, 2). It consists in soaking the cellswith a polymer, inducing the polymerization to form a dense meshworkthroughout the cell, cross-linking the fluorophores to the polymer and, afterdigestion of cellular protein, rehydrating of the sample. The swelling ofthe 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 sucha technique is the long preparation made of several stages, i.e. immunostaining,gelation, digestion and expansion. They are really crucial steps for a good im-aging post-expansion.In our work we present a comparison between ExM and stimulated emissiondepletion (STED) nanoscopy(3). Our aim is to study the e possible combinationof STED and ExM as a method to further enhance the final resolution achiev-able. We will in particularly take advantage of the use of separation of photonsby lifetime tuning (SPLIT) STED (4).We show application of these methods from single fixed cells to slices of fixedmouse retina tissue.We are also interested in applying the approach to high-density compartmentslike the cell nucleus to decipher the high-order structure organization of chro-matin-DNA
Combining Expansion Microscopy and STED Nanoscopy for the Study of Cellular Organization
Cainero I.;Pesce L.;Zanini G.;Lanzanò L.;Diaspro A.;Bianchini P
2017-01-01
Abstract
Expansion microscopy (ExM) is a novel method that allows super-resolutionimaging with conventional microscopes(1, 2). It consists in soaking the cellswith a polymer, inducing the polymerization to form a dense meshworkthroughout the cell, cross-linking the fluorophores to the polymer and, afterdigestion of cellular protein, rehydrating of the sample. The swelling ofthe 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 sucha technique is the long preparation made of several stages, i.e. immunostaining,gelation, digestion and expansion. They are really crucial steps for a good im-aging post-expansion.In our work we present a comparison between ExM and stimulated emissiondepletion (STED) nanoscopy(3). Our aim is to study the e possible combinationof STED and ExM as a method to further enhance the final resolution achiev-able. We will in particularly take advantage of the use of separation of photonsby lifetime tuning (SPLIT) STED (4).We show application of these methods from single fixed cells to slices of fixedmouse retina tissue.We are also interested in applying the approach to high-density compartmentslike the cell nucleus to decipher the high-order structure organization of chro-matin-DNA
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