Ultra-high resolution structural and molecular imaging of cells and tissues

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Far-field fluorescence microscopy is a powerful tool in biological research due to its live cell compatibility and molecular specificity. A major hurdle over the last ~100 years has been the limited resolution due to the diffraction of light. Modern super-resolution microscopy methods such as single-molecule localization microscopy (SMLM) overcame this fundamental barrier and improved the resolution of fluorescence microscopy ten-fold by stochastically switching single dyes on and off such that their emission events are separated in time. This allows their center positions to be localized with high precision in space, leading to a reconstructed super-resolved image with a resolution down to ~25 nm. However, current developments and applications of SMLM focus on fixed cells in thin samples and cellular structures that lie close to the coverslip surface. Indeed, the profound impact of SMLM on biomedical studies has yet to fully unfold due to the following limitations: (1) live-cell SMLM is slow and difficult to achieve ultrahigh resolution due to the small photon budget, the insufficient information carried per photon, and the required high excitation power; (2) SMLM through large tissue depths remains difficult, due to the rapidly deteriorating resolution and image fidelity in tissue specimens caused by aberration and fluorescence background; and, (3) molecular resolution (1-5 nm) is yet achievable in whole cells and tissues at low photon flux conditions. Overcoming these hurdles will help reveal the structure, function and dynamics for cellular constituents at the molecular resolution in living specimens, and the reconstruction of nanoscale maps of multiple protein species within a large tissue volume. These capacities will drastically expand the impact of SMLM applications. Our long-term goal is to develop novel optical imaging systems that achieve significant advances in defining the structure and function of cellular constituents in live cells and tissues with molecular resolution. In the next five years, we will focus on two research directions: (1) We will develop novel single molecule super-resolution imaging technologies and a phase-encoded localization method to enable molecular-resolution 3D imaging in live cells under low photon flux conditions. The innovations will enable us to capture 3D dynamics with 1-5 nm resolution and construct time-evolved structural models of macromolecular assemblies in live cells. (2) We will develop novel instruments and analytical methods to allow ultra-high resolution, multiplexed mapping of fluorescently labeled targets in large tissue volumes. We will apply these developments to reveal the molecular organization and functions of networks of actin filaments and myosins during the formation and constriction of the cytokinetic contractile ring in live fission yeast. Also, we will determine the precise subcellular localization of molecular motors like dynein with respect to both microtubule and actin in neuronal growth cones. We will also explore the correlation between nanoscale topology of chromatin loci with defined epigenetic content and cell lineage and changes in gene expression profile.