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

远场荧光显微镜是生物研究的有力工具，因为它具有活细胞兼容性和 分子特异性。在过去的100年里，一个主要的障碍是由于绕射而导致的分辨率有限 光的力量。单分子定位显微镜(SMLM)等现代超分辨显微技术 克服了这一根本障碍，将荧光显微镜的分辨率提高了十倍 随机开启和关闭单一染料，以使它们的发射事件在时间上分开。这使得 它们的中心位置在空间上被高精度定位，导致重建的超分辨率 分辨率降至~25 nm的图像。 然而，目前SMLM的发展和应用主要集中在薄层样品和细胞中的固定细胞 靠近盖层滑动面的结构。事实上，SMLM对生物医学研究的深远影响 由于以下限制，尚未完全展开：(1)活细胞SMLM速度慢，难以实现超高 由于光子预算较小、每个光子携带的信息不足以及所需的高分辨率 激发功率；(2)由于SMLM的迅速恶化，通过大组织深度的SMLM仍然困难 由像差和荧光背景引起的组织样品的分辨率和图像保真度； 在低光子通量条件下，分子分辨率(1-5 nm)仍然可以在整个细胞和组织中实现。 克服这些障碍将有助于揭示细胞成分的结构、功能和动力学 活体样品的分子分辨率和多种蛋白质物种的纳米级图谱的重建 在一个大的组织体积内。这些能力将极大地扩大SMLM应用程序的影响。 我们的长期目标是开发新的光学成像系统，在定义 具有分子分辨率的活细胞和组织中细胞成分的结构和功能。在下一个 五年来，我们将重点研究两个方向：(1)开发新颖的单分子超分辨 实现分子分辨率三维成像的成像技术和相位编码定位方法 低光子通量条件下的活细胞。这些创新将使我们能够捕捉到1-5纳米的3D动态 解析并构建活细胞中大分子组装的随时间演变的结构模型。(2)我们会 开发新的仪器和分析方法，以实现超高分辨率、多路复用的地图绘制 大组织体积中的荧光标记靶标。 我们将利用这些进展来揭示肌动蛋白网络的分子组织和功能。 裂解酵母细胞动力收缩环形成和收缩过程中的细丝和肌球蛋白。 此外，我们还将确定像dynein这样的分子马达相对于两者的精确亚细胞定位。 神经元生长锥体内的微管和肌动蛋白。我们还将探索纳米级拓扑之间的相关性 染色质基因座具有明确的表观遗传含量和细胞谱系，以及基因表达谱的变化。

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.