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

单分子和超分辨率成像方法，在密集拥挤的环境中具有最大光子效率、更高的时空分辨率和高检测灵敏度

• 批准号: 10005376
• 负责人: Alexandros Pertsinidis
• 金额: $ 22.45万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005376
• 关键词: 3-Dimensional; Algorithms; Area; Biochemistry; Biological; Biological Process; Biomedical Research; Budgets; Cell Nucleus; Cells; Cellular Structures; Cellular biology; Complex; Crowding; DNA Polymerase II; Data; Detection; Developmental Biology; Diffuse; Discipline; Disease; Environment; Equilibrium; Event; Fluorescence; Fluorescent Probes; Genetic; Goals; Health; Image; Imaging Device; Immunology; In Situ; Individual; Interferometry; Label; Length; Macromolecular Complexes; Methods; Microscope; Microscopy; Molecular; Molecular Structure; Monitor; Movement; Neurosciences; Optics; Organism; Phase; Photobleaching; Photons; Process; RNA; Resolution; Sampling; Signal Transduction; Specificity; Specimen; Structure; Techniques; Technology; Three-Dimensional Imaging; Time; base; biological systems; experimental study; fluorescence imaging; fluorophore; genomic locus; imaging approach; imaging modality; in vivo; instrument; interest; knowledge base; macromolecular assembly; meetings; millisecond; molecular imaging; molecular scale; nanometer; new technology; novel; particle; photo switch; prototype; single molecule; spatiotemporal; structural biology; temporal measurement; three-dimensional visualization; tool; trafficking; transcription factor

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.

抽象的 对生物过程和机制有更全面的了解 健康和疾病的基础需要更好地整合多个领域的信息 长度和时间尺度。超分辨率显微镜和单分子方法 成为扩展现场空间分辨率和检测灵敏度的有效工具 生物成像。然而，当前最先进的技术通常只能实现有限的 3D 分辨率妨碍了分子尺度上空间组织的可视化。而且 平衡时间和空间分辨率之间的权衡，同时以有限的操作 光子预算通常会导致单分子观察时间严重缩短。最后， 当对来自单通道的微弱信号进行成像时，许多显微镜配置都面临着挑战。 分子，特别是由于拥挤的细胞样本中的高背景。因此，虽然 有前途的是，单分子/超分辨率方法的全部潜力可以改变我们的 对生物过程的分子理解尚未实现。填补关键技术 间隙，需要新的优化显微镜配置 - 可以在极限下运行 时空分辨率，同时最大化暗淡荧光信号的信息内容。 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， 有针对性的荧光切换，同时进一步利用新兴的光子高效算法 提高分辨率并延长总观察时间。基于这些想法我们 建议开发新型超分辨率和单分子荧光成像工具， 重点关注两个具体目标：（1）扩展基于本地化的时空尺度 单分子成像和跟踪可达 1 纳米各向同性 3D 分辨率和 ~1,000 数据点体内观察轨迹的采样率低至（亚）毫秒； (2) 实现 在存在微量元素的情况下，可寻址 3D 体积中的实时单分子检测灵敏度 摩尔背景浓度，以及高度拥挤的细胞内环境。这 新技术将显着提高我们探究动态生物的能力 具有分子细节的过程，从而对整个领域产生广泛而直接的影响 生物医学学科。