New Dimensions and Domains for Fluorescence Fluctuation Imaging Methods

在过去的十年里，光学显微镜领域发生了一场革命，2014年化学诺贝尔奖授予了Betzig， Moerner和Hell，以表彰他们开发超分辨荧光显微镜。这一技术复兴见证了超分辨率荧光显微镜和单平面照明显微镜（SPIM）也被称为光片显微镜的巨大进步。就像17世纪光学显微镜的出现一样，这些新的技术飞跃为活细胞和生物体的生物医学研究提供了巨大的希望。然而，许多形式的超分辨率显微镜是基于迭代的分子定位，导致动力学瓶颈，牺牲时间分辨率来获得空间分辨率。***本提案旨在通过开发适用于超分辨率和薄片显微镜方法的新型定量荧光波动分析工具来弥补这一差距。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，尤其是在后基因组时代，生物学需要向新的定量基础发展。NSERC-DG的资助对我们开发新的生物物理图像分析工具至关重要，这些工具可以解开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一种对图像中荧光波动的噪声分析，该分析报告了细胞中被成像的分子的行为。通过在空间和时间上关联信号，我们可以揭示荧光标记分子的移动速度，无论它们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，包括多尺度的小波分析，特别适合于处理在更现实的模型3D系统和生物体组织中生长的细胞遇到的更困难的细胞形状。该建议的重点是扩展我们的图像相关性，荧光波动和小波工具，以测量超分辨率显微镜测量的生物分子动力学，并在细胞中受体循环的3D应用中进行测量，最后在3D中测量胶原酶动力学图，用于模型癌细胞在更真实的3D胶原基质中迁移。该DG计划建立在定量荧光方法开发，显微镜/光学以及3D光/晶格片显微镜和所有主要形式的超分辨率显微镜的全新实验室仪器方面表现卓越的坚实基础上。

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.