Evaluating the effect of different Expansion Microscopy protocols on mammalian DNA-Chromatin architecture

Expansion microscopy (ExM) is a novel super-resolution microscopy method that enables nano-scale biophysical studies by increasing the distance among biomolecules. Such effect is achieved through the physical magnification of the specimen obtained by its embedding inside a swellable polymer network, eventually expanded in water. Several improvements to the original ExM technique were made to optimize the imaging of proteins and the isotropicity of their expansion. However, few works have been done to investigate what happens to the DNA-chromatin architecture once it’s subjected to the mechanical strain associated with the expansion process. To understand whether this technique could be suitable for investigating the crowded nuclear environment, we aim to study the induced changes in the DNA conformation by the application of different expansion microscopy protocols to fixed mammalian cells and single mononucleosomes. Optical imaging will be performed using a combination of confocal, two-photon excitation and super-resolution microscopy techniques, and the performances of different fluorophores for DNA staining will also be compared. Finally, we aim to develop a reproducible protocol for the precise quantification of the expansion factor of the specimens at the microscale.