Single-molecule and super-resolution imaging methods with maximum photon efficiency, increased spatiotemporal resolution and high detection sensitivity in densely crowded environments

摘要 对生物过程和机制有更全面的了解 基础健康和疾病要求更好地集成跨越多个 长度和时间刻度。超分辨率显微镜和单分子方法已经 成为扩展LIVE空间分辨率和检测灵敏度的有力工具 生物成像。然而，当前最先进的技术通常实现有限的3D 无法在分子尺度上可视化空间组织的分辨率。更有甚者 平衡时间和空间分辨率之间的权衡，同时在有限的 光子预算往往会导致单分子观测时间严重缩短。最后， 许多显微镜配置在成像来自单个显微镜的微弱信号时受到挑战。 分子，特别是由于拥挤的细胞样本中的高背景。因此，虽然 有希望的是，单分子/超分辨率方法的全部潜力将改变我们的 对生物过程的分子理解还没有实现。填补关键的技术问题 间隙，需要新的优化显微镜配置-可以在以下限制下运行 时空分辨率，同时最大化微弱荧光信号的信息量。 我们假设这一目标可以通过3D干涉测量的新颖组合来实现， 定向荧光开关，同时进一步利用新兴的光子效率算法 以提高分辨率并延长总观测时间。基于这些想法，我们 建议开发新的超分辨率和单分子荧光成像工具， 针对两个具体目标：(1)扩展基于定位的时空尺度 单分子成像和跟踪达到1纳米各向同性3D分辨率和~1,000 数据点在体内的观察轨迹低至(亚)毫秒的采样率；(2)实现 实时单分子检测灵敏度在可寻址的3D体积中，在存在微 摩尔背景浓度，以及在高度拥挤的细胞内环境中。这个 新技术将显著提高我们审问动态生物的能力 具有分子细节的过程，从而对 生物医学学科。

ABSTRACT Reaching a more complete understanding of biological processes and mechanisms that underlie health and disease demands a better integration of information spanning multiple length and time scales. Super-resolution microscopy and single-molecule approaches have emerged as potent tools that extend the spatial resolution and detection sensitivity in live biological imaging. However, the current state-of-the-art techniques often achieve limited 3D resolution that precludes visualizing spatial organization at the molecular scale. Moreover balancing trade-offs between temporal and spatial resolution, while operating with a limited photon budget often results in severely shortened single-molecule observation times. Finally, many microscope configurations are challenged when imaging weak signals from single- molecules, especially due to high background in crowded cellular specimens. Thus, although promising, the full potential of single-molecule/super-resolution methods for transforming our molecular understanding of biological processes has yet to be realized. To fill critical technical gaps, new optimized microscope configurations are needed - that can operate at the limits of spatiotemporal resolution while maximizing the information content of dim fluorescence signals. We hypothesize that this goal can be achieved through novel combinations of 3D interferometry, targeted fluorescence switching, while further harnessing emerging photon-efficient algorithms to increase resolution as well as prolong total observation times. Based on these ideas we propose to develop novel super-resolution and single-molecule fluorescence imaging tools, focusing on two specific aims: (1) To extend the spatiotemporal scales of localization-based single-molecule imaging and tracking to 1 nanometer isotropic 3D resolution and to ~1,000 data-point in vivo observation traces at down to (sub)millisecond sampling rates; (2) To achieve real-time single-molecule detection sensitivity in addressable 3D volumes, at presence of micro- Molar background concentrations, and inside highly crowded intracellular environments. The new techniques will significantly increase our abilities to interrogate dynamic biological processes with molecular detail, thus having widespread and immediate impact across biomedical disciplines.