Technical Advances in Spectroscopic Single-Molecule Localization Microscopy for Improving Spatial Resolution and Spectral PrecisionPublic
Fluorescence microscopy has become a widely used tool in many research areas. However, its spatial resolution, limited to 250 nm by the diffraction limit of light, has restricted direct observation of details of ultrastructural biology. In recent years, spectroscopic single-molecule localization microscopy (sSMLM), one of super-resolution imaging techniques, has been recognized as a very powerful tool to offer molecular insights into cellular behavior. It enables the visualization of nanoscopic features of cells far beyond the diffraction limit of the conventional fluorescence microscopy by capturing spatial information of fluorescent molecules, achieving spatial resolution on the order of 40-80 nm. Furthermore, this technique has been attracting significant interest by additionally capturing the linked spectroscopic signatures. However, the sSMLM system has an intrinsic constraint imposed by the limited photon budget of individual molecules as the emitted photons need to be separated into two imaging domains to capture spatial and spectral images simultaneously. This fundamental constraint restricts the highest level of spatial resolution and spectral precision in sSMLM and thus hampers the potential for various structural/functional imaging applications. This dissertation has provided technical advances in sSMLM to address this issue. The work in this dissertation covers a range of topics under this overall goal: (1) theoretical studies to evaluate the performance of sSMLM using numerical simulation and analytical solution; (2) development of new optical configurations and post-data processing techniques to improve spatial and spectral precisions. Specifically, four technical advances are presented: (i) tunable spectral dispersion sSMLM, (ii) three-dimensional (3D) biplane sSMLM, (iii) symmetrically dispersed sSMLM (SDsSMLM), and (iv) photon-accumulation enhanced reconstruction (PACER); (3) their experimental validations for multimodal imaging using biology samples and nanoparticles; and (4) Manufacture of a compact optical device integrating all demonstrated 3D sSMLM functionalities. These studies offer a comprehensive technical guidance for better designing and optimizing the sSMLM system, which can facilitate more precise spectroscopic single-molecule study in broader cell biology and material science applications.