Synthesis and Evaluation of novel Multifunctional Labels for Expansion Microscopy

Expansion microscopy (ExM), a relatively new super-resolution imaging method, enables visualization of biological targets at nanoscale resolution on diffraction limited microscopes. Since its invention in 2015, ExM has aroused a tremendous attention and various ExM variants have been developed to simplify its usage and increase the imaging resolution that can be achieved. However, through common biomolecule-grafting methods, ExM is limited to some biomolecules, such as proteins and nucleic acids. In addition, fluorescence loss is inevitable during radical-induced polymerization and digestion due to the incomplete anchoring of fluorescent labels. In this thesis, to solve these limitations, we design and synthesize novel multifunctional molecules.

In chapter 1, we provide a general introduction to ExM. We summarize the current progress of ExM, with an emphasis on the chemical aspects of the method. In addition, we discuss the strategies applied for additional resolution improvement via combining ExM with different super-resolution microscopes.

In chapter 2, the limitations of commercially available fluorescent labels are analyzed when applied in ExM and the goals of this thesis are demonstrated.

In chapter 3, the direct grafting strategy of fluorescent labels is evaluated via synthesizing a series of TRITON molecules. These molecules enable simultaneous targeting, labeling and grafting of various biological targets. The first example of lipid membrane and actin filament staining is demonstrated in ExM. In addition, a post-labeling strategy is validated via introducing readout probes post expansion in oligonucleotide-mediated immunofluorescence.

In chapter 4, an optimized grafting strategy of actin filaments is described. A series of improved TRITON molecules with multiple acryloyl units is synthesized. The combination of our optimized phalloidin TRITON with additional anchoring reagents is demonstrated to enable a significant improvement of the fluorescent signal retention of phalloidin TRITON.

In chapter 5, a universal labeling strategy for nucleic acids is described. A series of novel di/multivalent molecules is synthesized to allow rapid functionalization of DNA oligonucleotides as probes for expansion microscopy, in a single approach. These reagents are compatible with third-generation in situ hybridization chain reaction RNA-FISH techniques and multi-color staining. The TRITON-functionalized oligonucleotides also allow the immunofluorescence staining of microtubules using different labeling strategies.

In chapter 6, impact of photostabilizers on radical-induced fluorophore damage is examined in ExM. Photostabilizer-conjugated organic fluorophores show an obviously improved fluorescent signal retention during the radical-induced polymerization step. Two strategies are explored to allow the use of photostabilizers in ExM. Through direct conjugation of photostabilizers with our TRITON molecules, an improved fluorescent intensity and photostability of dyes is demonstrated after expansion. Furthermore, a series of photostabilizer-modified anchoring reagents is synthesized to extend the application of this finding to commercially available fluorophore-conjugated antibodies.

Finally, a general conclusion of the work is given and based on our observations and findings, a few directions for future work are discussed in chapter 7.