Novel applications of and developments in expansion microscopy.

Novel applications of and developments in expansion microscopy. The field of super-resolution microscopy is quickly expanding, and one of the latest methods in this field is expansion microscopy (ExM). Instead of using optics and advanced imaging schemes such as in PALM, STORM or STED, ExM makes use of the physical enlargement of samples to bypass the resolution limit. Cells or tissues are embedded in a polymer gel which is subsequently expanded isotropically by dialysis with water. This expansion increases the distances between molecules 4-10-fold and this process effectively increases the resolution 4-10 fold. It has a clear advantage compared to other super-resolution techniques, namely that it does not require any special hardware, software or dyes, which makes it accessible for many biological labs. Indeed, since the method’s inception in 2015, it has been widely adopted. In this thesis, we want to push the boundaries of ExM by technical developments and by identifying novel applications. One such application comprises single-cell multi-omics technologies, where ExM is expected to support the development of high throughput methods. Single cell studies make use of fluorescent probes that sequentially bind to various biomolecules. By increasing the resolution, the number of marked biomolecules that can be detected will substantially increase. A second target will be the use of ExM in membrane studies, the small elusive lipid rafts associated within them. We also want to apply ExM, for the first time, to study focal adhesions. These are structures the cell uses to attach to its surroundings. Although these are easily detected in 2D cell cultures, the study of focal adhesions for cells in a 3D environment is much more challenging. Using expansion microscopy to expand the 3D environment may reveal more information about these focal adhesions. Finally, ExM is a technique that is very suitable to be combined with other super-resolution techniques, with the potential to achieve real molecular resolution. We will explore the combination of STED and ExM through specific developments of the laboratories proprietary Triton linker.