New Dimensions and Domains for Fluorescence Fluctuation Imaging Methods

荧光波动成像方法的新维度和新领域

• 批准号: RGPIN-2017-05005
• 负责人: Wiseman, Paul
• 金额: $ 7.72万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=668734
• 关键词: New; Dimensions; Domains; Fluorescence; Fluctuation

Advancing quantitative image correlation methods to the third dimension and beyond***In the past decade there has been a revolution in light microscopy recognized with the awarding of the 2014 Chemistry Nobel Prize to Betzig, Moerner and Hell for the development of super resolved fluorescence microscopy. This technical renaissance has witnessed tremendous advances in both super-resolution fluorescence microscopy and in single plane illumination microscopy (SPIM) also commonly known as light sheet microscopy. As was the case at the advent of light microscopy in the 17th century, these new technological leaps offer great promise for biomedical research science on living cells and organisms. However, many forms of super-resolution microscopy are based on iterative molecular localization leading to a kinetic bottleneck where temporal resolution is sacrificed to gain spatial resolution. ***This proposal is based on bridging this gap by developing new quantitative fluorescence fluctuation analysis tools suitable for super-resolution and light sheet microscopy methods. If a picture is worth a thousand words, a quantitative understanding of the dynamics of the molecular machines of life inside the cell is even more valuable especially as Biology needs to move toward a new quantitative basis in the post Genomic era. NSERC-DG funding has been fundamental in allowing us to develop new biophysics image analysis tools that unlock information on the transport properties and interactions of molecules imaged with standard fluorescence microscopy methods. These image correlation methods rely on a type of noise analysis of fluorescence fluctuations in the images which report on how the molecules imaged in the cell are behaving. By correlating the signals in space & time, we can reveal how quickly the fluorescently tagged molecules are moving, whether they are diffusing randomly or moving in a directed fashion, measure numbers of molecules in a given state, and report on the binding & interactions of molecules. We have also extended our method toolkit to include wavelet analysis at multiple scales which is especially suited for tackling the more difficult cell shapes encountered for cells growing in more realistic model 3D systems and in the tissues of living organisms. This proposal is centered on extending our image correlation, fluorescence fluctuation and wavelet tools to measure biomolecule dynamics from super-resolution microscopy measurements and in 3D applications for receptor cycling in cells and finally to measure collagenase enzyme kinetics maps in 3D for model cancer cells migrating in more realistic 3D collagen matrices. This DG program is built on a firm foundation of demonstrated excellence in quantitative fluorescence method development, microscopy/optics and completely new lab instrumentation in 3D light/lattice sheet microscopy and all major forms of super-resolution microscopy.

在过去的十年里，光学显微镜领域发生了一场革命，2014年诺贝尔化学奖授予Betzig、Moerner和Hell，以表彰他们对超分辨荧光显微镜的发展。这场技术复兴见证了超分辨率荧光显微镜和单面照明显微镜(SPIM)的巨大进步，SPIM也通常被称为光片显微镜。就像17世纪光学显微镜出现时一样，这些新的技术飞跃为活细胞和生物的生物医学研究科学提供了巨大的希望。然而，许多形式的超分辨率显微镜是基于迭代分子定位的，这导致了动力学瓶颈，其中牺牲了时间分辨率来获得空间分辨率。*这项建议是基于通过开发适用于超分辨率和光片显微镜方法的新的定量荧光波动分析工具来弥合这一差距。如果一张图片胜过千言万语，那么对细胞内生命分子机器动力学的定量理解就更有价值了，特别是在生物学需要在后基因组时代走向新的量化基础的情况下。NSERC-DG的资金一直是使我们能够开发新的生物物理图像分析工具的基础，这些工具解锁了用标准荧光显微镜方法成像的分子的传输特性和相互作用的信息。这些图像相关方法依赖于对图像中荧光波动的一种噪声分析，该分析报告了细胞中成像的分子的行为方式。通过在空间和时间上关联信号，我们可以揭示荧光标记的分子移动的速度有多快，无论是随机扩散还是定向移动，测量给定状态下的分子数量，并报告分子的结合和相互作用。我们还扩展了我们的方法工具包，包括多个尺度的小波分析，这特别适合于处理在更逼真的3D模型系统中生长的细胞和在生物组织中生长的细胞所遇到的更困难的细胞形状。这项建议的核心是扩展我们的图像相关性、荧光波动和小波工具，以从超分辨率显微镜测量和在细胞内受体循环的3D应用中测量生物分子动力学，并最终测量胶原酶在3D中迁移到更逼真的3D胶原基质中的模型癌细胞的胶原酶动力学图。这个DG项目建立在一个坚实的基础上，该项目在定量荧光方法开发、显微镜/光学和3D光学/晶格片显微镜以及所有主要形式的超分辨率显微镜方面展示了全新的实验室仪器。