UC Davis

With the advancement in the super-resolution microscopy technique, the resolving power of the microscope can surpass the accuracy provided by conventional labeling method. Positioning reporter molecules as close as possible to the target protein becomes the main challenge in super-resolution imaging. Therefore, the aim of this study is to showcase the resolution improvement in super-resolution microscopy when using peptide based small molecule probes.In Chapter I, I will provide an overview of this thesis. I will start with a general description of the current challenges in microscopic imaging and why it is becoming more important for developing new small-molecule probes that provides a better accuracy than developing new super-resolution imaging techniques to push the resolution boundaries. Next, I will discuss different screening methods for generating probes and the limitation they are facing, which lead to the idea of using one-bead one-compound (OBOC) library screening in combination of a fluorogenic reporter, fluorescent molecular rotor (FMR) to build a novel library screening platform that has high-throughput capability for screening, defined chemical structure for reproducibility, fluorogenic property suitable for live imaging, and most important of all, having a small molecule size to reduce the linkage error in super-resolution microscopy. Although I did not have a solid sequence obtained from this library, I use a peptide probe, eSylite, derived from a known binding sequence to demonstrate the idea that small molecule size probes can provide a better apparent size in labeling by using super-resolution imaging. In Chapter II, I will provide the detail discussion and results from my FMR-OBOC library experiments and the reason why I concluded it lacks the sensitivity for screening. Though the attempts did not end up with a fruitful of results, I came up with the idea of using fluorescence lifetime instead of fluorescence intensity as an alternative parameter for screening that can possibly avoid issues that jeopardize the previous screening design. In Chapter III, I will discuss a novel fluorescence peptide probe, eSylite, designed to have nano-molar affinity and high specificity toward the scaffolding PSD-95 protein, a key marker for excitatory postsynaptic density. I will demonstrate, using different microscopy methods including confocal laser scanning microscopy, Stimulated Emission Depletion (STED) microscopy, and direct Stochastic Optical Reconstruction Microscopy (dSTORM), that the eSylite probe provides high specificity for PSD-95 only, without cross-reactivity with other members of the same family. The small peptide probe yields a smaller apparent size in imaging, indicating reduced linkage error in pinpointing the true location of PSD-95, as well as improved resolution, enabling the visualization of isolated nanodomain structures of PSD-95 compared to the traditional antibody labeling method. In summary, this thesis provides the design and attempts to use FMR-OBOC strategy as a novel screening platform in seek of fluorogenic small molecule peptide probes for super-resolution imaging. Despite the failure in this library screening method, I used a small molecule peptide probe derived from a known sequence to demonstrate the downsizing of probes can provide better labeling accuracy and better resolution in super-resolution imaging.