IDBR: Super-Resolution Made Super-Easy via (Transient-)PhILM

IDBR：通过（瞬态）PhILM 使超分辨率变得超级简单

• 批准号: 1063188
• 负责人: Paul Selvin
• 金额: $ 51.63万
• 依托单位: University of Illinois at Urbana-Champaign
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1063188&HistoricalAwards=false
• 关键词: IDBR; Super; Resolution; Made; Easy

Project AbstractRecently the biological imaging community has seen tremendous improvements in imaging resolution. Super-resolution fluorescence microscopy techniques, such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), have enabled imaging at resolutions approaching 20 nm on fixed cells, much below the typical ~200-nm resolution of conventional microscopy. Furthermore, microscope companies, including Nikon, Zeiss, and Leica, have each introduced super-resolution microscopes. However, these techniques require a number of specialized optics, specialized dyes, and generally have difficulty with living cells. What would be incredibly valuable to the biological community would be a "simple" technique such that they could use a microscope?possibly their own, perhaps with a little "tweaking"--and use their own labels, not necessarily at the strictly single-molecule level, but still achieve "super-resolution". In addition, the PI's group has recently shown another super-accuracy technique--it is possible to get 3-nm accuracy in all three dimensions by two-photon microscopy of quantum dots. In this project, both the "simple" 1-photon and more challenging 2-photon super-resolution microscopy methods will be exploited. Intellectual Merit: Two related microscopy techniques, one called Photobleaching and Intermittency Localization Microscopy (PhILM) and a close cousin, Transient-PhILM, will be developed using commercially available high-resolution cameras and fluorophores. For initial testing, a special Nikon microscope, built for the PI while he was a Nikon Fellow at the Marine Biological Laboratory in Summer 2010, will be used. Methods to transfer the technology to common microscopes will then be undertaken.The idea behind PhILM is to label a molecular structure--for example, a microtubule--with many dyes and then photobleach them one at a time. The images are recorded sequentially and then played backwards. For example, if an image with n-1 fluorophores is taken and subtracted from the image with n fluorophores, the image of a single dye molecule is obtained. Using fluorescence imaging with one-nanometer accuracy (FIONA) fitting, the centroid can be obtained with nanometer accuracy. The analysis is then repeated with the image having n-2 fluorophores subtracted from the n-1 fluorophore image, yielding nanometer accuracy again. Thus, a super-resolution image of many fluorophores is achieved. If too many fluorophores are present, a discrete step may not be observable. However, by simply exciting until a sufficient number of fluorophores have photobleached, subsequent individual events may be observed. Because the subtraction scheme introduces extra photon noise (due to surrounding fluorophores), it will likely be less sensitive than PALM or STORM. However, the wide applicability with existing microscopes makes the technique attractive. Improvements to the technology--dealing with microscope drift, chromatic aberrations of the objective, testing with GFPs and blinking quantum dots, making soft-ware to automate the procedure, and testing the technique on nucleic acids, will be undertaken in this project. The second technique to be developed is Transient PhILM. Here, a biomolecule of interest is labeled with a fluorophore, such that the dye is attached for only a "brief" period of time. During this time, the fluorophore is stationary and emits intensely at one spot. The spot can be localized via FIONA to give its centroid to a sub-diffraction limited size, usually a few nanometers. When the fluorophore is not attached, it floats freely and contributes a haze, which has been shown to be insignificant in most cases. Then the dye, whether photobleached or not, floats away and another dye binds, and the procedure is repeated. This method could potentially have better resolution than PALM or STORM, which are limited by the number of times a dye-pair can be turned on and off. The trick is to have the dye remain bound for a "brief" period of time. In preliminary results, this technique is shown to be feasible. Broader Impacts: The potential for commercialization of this technology is significant. The PI has two patent applications submitted, and Nikon has expressed interest in commercializing the technology. Two companies have expressed interest in modifying their reagents for Transient PhILM. The PI is a principal with the NSF-funded Physics Frontiers Center, Center for the Physics of Living Cells (CPLC) at Illinois. In addition to the students being trained directly on this project, the PI participates in the annual CPLC summer school, which typically trains 40 to 50 advanced graduate students and postdocs in single-molecule techniques. Nanohub.org, an NSF-funded web-site, will disseminate the software produced in this project. Finally, the work will be done by graduate students, including four women currently supervised by the PI.

项目摘要最近，生物成像界已经看到了成像分辨率的巨大进步。超分辨率荧光显微镜技术，如光激活定位显微镜（PALM）和随机光学重建显微镜（STORM），已经能够在固定细胞上以接近20 nm的分辨率成像，远低于传统显微镜的典型~200 nm分辨率。此外，显微镜公司，包括尼康，蔡司和徕卡，都推出了超分辨率显微镜。然而，这些技术需要许多专门的光学器件、专门的染料，并且通常难以处理活细胞。对生物界来说，最有价值的是一种“简单”的技术，比如他们可以使用显微镜？也许是他们自己的，也许是一个小的“调整”-并使用他们自己的标签，不一定在严格的单分子水平，但仍然实现“超分辨率”。此外，PI的团队最近展示了另一种超精度技术-通过量子点的双光子显微镜可以在所有三维空间中获得3 nm的精度。在这个项目中，“简单”的单光子和更具挑战性的双光子超分辨率显微镜方法都将被利用。智力优势：两个相关的显微镜技术，一个称为光漂白和间歇定位显微镜（PhILM）和一个近亲，瞬态PhILM，将开发使用商业上可用的高分辨率相机和荧光团。对于初始测试，将使用一台特殊的尼康显微镜，这是PI在2010年夏天担任海洋生物实验室尼康研究员时为他制造的。PhILM背后的想法是用许多染料标记一个分子结构--例如，一个微管--然后一次光漂白它们中的一个。图像按顺序记录，然后向后播放。例如，如果拍摄具有n-1个荧光团的图像并从具有n个荧光团的图像中减去，则获得单个染料分子的图像。利用一纳米精度的荧光成像（FIONA）拟合，可以获得纳米精度的质心。然后用从n-1个荧光团图像中减去n-2个荧光团的图像重复分析，再次产生纳米精度。因此，实现了许多荧光团的超分辨率图像。如果存在太多的荧光团，则可能无法观察到离散步骤。然而，通过简单地激发直到足够数量的荧光团已经光漂白，可以观察到随后的个体事件。由于减法方案引入了额外的光子噪声（由于周围的荧光团），因此它可能不如PALM或STORM敏感。然而，现有显微镜的广泛适用性使该技术具有吸引力。该项目将对该技术进行改进-处理显微镜漂移、物镜色差、用玻璃纤维膜和闪烁量子点进行测试、制作软件使程序自动化以及在核酸上测试该技术。第二种技术是瞬态PhILM。在这里，感兴趣的生物分子用荧光团标记，使得染料仅附着“短暂”的时间段。在此期间，荧光团是固定的，并在一个点强烈发射。光斑可以通过FIONA定位，以使其质心达到亚衍射极限尺寸，通常为几纳米。当荧光团没有附着时，它自由漂浮并产生雾度，这在大多数情况下都是微不足道的。然后染料，无论光漂白与否，漂走，另一种染料结合，重复这个过程。这种方法可能具有比PALM或STORM更好的分辨率，PALM或STORM受到染料对可以打开和关闭的次数的限制。诀窍是让染料保持“短暂”的结合时间。初步结果表明，该技术是可行的。更广泛的影响：这项技术的商业化潜力巨大。PI已经提交了两项专利申请，尼康表示有兴趣将该技术商业化。两家公司表示有兴趣修改其用于瞬时PhILM的试剂。PI是NSF资助的物理前沿中心，伊利诺伊州活细胞物理中心（CPLC）的负责人。除了直接对学生进行该项目的培训外，PI还参加了一年一度的CPLC暑期学校，该学校通常培训40至50名高级研究生和单分子技术博士后。Nanohub.org是国家科学基金会资助的一个网站，将传播该项目制作的软件。最后，这项工作将由研究生完成，其中包括目前由PI监督的四名妇女。