Super-resolution microscope with fluorescence fluctuation and expansion gel imaging capabilities

具有荧光波动和膨胀凝胶成像功能的超分辨率显微镜

• 批准号: 524798474
• 金额: --
• 依托单位: Georg-August-Universität Göttingen
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2023
• 资助国家: 德国
• 起止时间: 2022-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524798474?language=en
• 关键词: Super; resolution; microscope; fluorescence; fluctuation

Fluorescence microscopes have been limited for decades by the diffraction barrier, to approximately half of the wavelength of the imaging light (200-350 nm in most biological experiments). Several super-resolution approaches have been developed to overcome the diffraction barrier, but fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Combining optical super-resolution technologies with expansion microscopy, in which the sample is enlarged after embedding in a swellable gel, should, in principle, reach molecular resolution. This failed, as the gels limited the effectiveness of most super-resolution tools, including both coordinate-targeted approaches (as STED or SIM) and single-molecule based approaches (as STORM). We recently obtained a solution to this problem, employing a third class of optical super-resolution approaches, which is based on determining the higher-order statistical analysis of temporal fluctuations measured in a movie. We combined expansion microscopy with a super-resolution radial fluctuations (SRRF) analysis and obtained 0.8 to 1 nm resolutions across different samples and color channels. We applied this technique, which we termed one-nanometer expansion microscopy (ONE) to many issues, from diagnostics to the analysis of the shape of single molecules. ONE microscopy opens many of new avenues in biological sciences, from an all-optical analysis of protein structure to various combinations of live super-resolution imaging and structural analyses. Higher performance, to resolutions substantially better than 1 nm, is possible, since the current results are only limited by the signal-to-noise ratio. To achieve this, we apply here for a microscope that will replace the setup on which we established ONE microscopy, while offering substantial advantages. In addition, we require the same setup for many other projects, dealing especially with living samples, to replace the general functionality of our previous setup. In short, we require a setup that will perform the following: 1) provide a dynamic analysis of the expanded gels, with excellent speed and signal-to-noise ratio, to obtain molecular-scale resolution; 2) allow the routine testing of the samples at super-resolution (50 nm or better), both in fixed and living cells; 3) enable sample analysis procedures as fluorescence recovery after photobleaching (FRAP); 4) be compatible with approaches that enable strong multiplexing, e.g. by use of fluorescence lifetime imaging. The required setup will be placed in the Biomedical Microscopy Unit of the University Medical Center Göttingen, and will replace a heavily used microscope installed in 2007, which will no longer be fully operational after 2023.

数十年来，荧光显微镜被衍射屏障限制了，大约是成像光的波长的一半（在大多数生物学实验中200-350 nm）。已经开发出了几种超分辨率方法来克服衍射屏障，但是荧光显微镜仍然无法成像单蛋白或小分子复合物的形态，即纯化或在细胞环境中。将光学超分辨率技术与膨胀显微镜相结合，其中嵌入在膨胀凝胶中后的样品应增加，原则上应达到分子分辨率。这失败了，因为凝胶限制了大多数超分辨率工具的有效性，包括坐标为目标的方法（如steed或sim）和基于单分子的方法（如风暴）。最近，我们采用了第三类光学超分辨率方法来解决该问题的解决方案，该方法基于确定电影中测得的临时波动的高阶统计分析。我们将膨胀显微镜与超分辨率径向波动（SRRF）分析相结合，并在不同的样品和颜色通道上获得了0.8至1 nm的分辨率。我们应用了这项技术，我们将其称为一纳米扩展显微镜（一个）到许多问题，从诊断到分析单分子的形状。一个显微镜开辟了生物科学的许多新途径，从蛋白质结构的全光分析到实时超分辨率成像和结构分析的各种组合。由于目前的结果仅受信噪比的限制，因此更高的性能要大于1 nm。为了实现这一目标，我们在这里申请了一个显微镜，该显微镜将取代我们建立一个显微镜的设置，同时提供实质性的优势。此外，我们需要为许多其他项目进行相同的设置，尤其是与活样本交易，以替换我们以前的设置的一般功能。简而言之，我们需要一个将执行以下操作的设置：1）对扩展的凝胶的动态分析，具有出色的速度和信噪比，以获得分子尺度的分辨率； 2）允许在固定细胞和活细胞中以超分辨率（50 nm或更高）进行样品的常规测试； 3）启用样品分析程序作为光漂白后的荧光恢复（FRAP）； 4）与能够强大的多路复用的方法兼容，例如通过使用荧光寿命成像。所需的设置将放置在大学医学中心Göttingen的生物医学显微镜单元中，并将取代2007年安装的大量使用的显微镜​​，该显微镜将在2023年之后不再完全运行。