Mechanistic Insights and Improvements in Expansion Microscopy

Abstract

Super-resolution microscopy enables the observation of structures below the diffraction limit of light. A relatively new method, Expansion Microscopy (ExM) involves physically expanding a hydrogel-embedded facsimile of a biological structure by tearing it apart. The resultant larger replica permits sub-diffraction imaging with a typical fluorescent microscope. However, there are challenges, such as retaining the fluorescent signal after expansion and the isotropic nature of the expansion procedure, which this study will address. The signal loss is due to the loss of labels, which is mainly caused by gel formation and digestion. To address this issue, an agent was developed in this study that combines targeting, fluorescent labeling, and gel linkage into a single small molecule. The findings presented here suggest that label loss is due to inadequate surface grafting of fluorophores into the hydrogel. In order to gain a mechanistic understanding of dye retention, it is crucial to understand the function of surface grafting in ExM. By delivering monomers directly to the target of interest via nanobodies, the amount of polymerizable units bound to the surface was increased, resulting in retention of the signal. Consequently, the density of local monomers is crucial for retaining signals. In addition to signal retention, it is essential to verify that the expansion was uniform in all directions, i.e. isotropic, since only a facsimile of the original biological structure is detected after expansion. This is necessary to draw accurate conclusions from the results. Therefore, my goal was to develop a ruler that measures the isotropy of the expansion. For this experiment, tobacco mosaic virus (TMV) particles were fluorescently labeled and introduced as an internal standard into cells by coupling them with microtubules. However, the distribution of particle lengths and the loss of signal after expansion proved to be problematic. These obstacles can be resolved in future research. A uniform virus length could be achieved through size exclusion chromatography. Secondly, signal retention could be improved by fluorescently labeling additional amino acids of TMV coat proteins (CPs) and providing particles with polymerizable groups.