![]() However, resolution alone is not sufficient for determining structure equally important is the accuracy with which the structural measurements can be made 11. Studies combining ExM with structured illumination microscopy (SIM) 3, 4, stimulated emission depletion (STED) microscopy 5โ 7, and stochastic optical reconstruction microscopy (STORM) 8โ 10 have already demonstrated the use of ExM to increase the resolution of super-resolution methods. Combination of these methods with expansion microscopy (ExM) 2 holds the potential to improve imaging capabilities further, through embedding the sample in a hydrogel followed by physical expansion of the gel. Super-resolution optical microscopy methods 1 have made it possible to investigate structural features on length scales smaller than the diffraction limit of light. Our study thus provides direct measurements of the single-molecule distortions resulting from hydrogel embedding, and presents a way to improve super-resolution microscopy through combination with tetra-gel ExM. By combining tetra-gel ExM with STORM, we were able to resolve 11-nm structural features without the loss in accuracy seen with polyacrylamide gels. In contrast, we found that embedding in tetra-gel, a hydrogel that does not depend on free-radical chain-growth polymerization, preserved labeled sites with a standard deviation of less than 5 nm. We found that embedding in hydrogels based on polyacrylamide, the most widely used chemistry in ExM, resulted in random displacements of labeled sites with a standard deviation ofโ~โ16 nm. ![]() Here, we quantified the accuracy of gel embedding by using stochastic optical reconstruction microscopy (STORM) to image DNA origami with well-defined structures. However, it has been unknown to what extent gel embedding alters the molecular positions of individual labeled sites. The accuracy of expansion microscopy (ExM) depends on the structural preservation of samples embedded in a hydrogel.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |