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.

几十年来，荧光显微镜一直受到衍射屏障的限制，大约是成像光波长的一半（在大多数生物实验中为200-350 nm）。已经开发了几种超分辨率方法来克服衍射障碍，但荧光显微镜仍然无法成像单个蛋白质或小分子复合物的形态，无论是纯化的还是在细胞环境中。将光学超分辨率技术与膨胀显微镜相结合，在膨胀显微镜中，样品在包埋在可溶胀的凝胶中后被放大，原则上应该达到分子分辨率。这失败了，因为凝胶限制了大多数超分辨率工具的有效性，包括坐标靶向方法（如STED或SIM）和基于单分子的方法（如STORM）。我们最近获得了这个问题的解决方案，采用第三类光学超分辨率方法，这是基于确定在电影中测量的时间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中获得了0.8至1 nm的分辨率。我们将这种称为一纳米膨胀显微镜（ONE）的技术应用于许多问题，从诊断到单分子形状的分析。ONE显微镜在生物科学中开辟了许多新的途径，从蛋白质结构的全光学分析到实时超分辨率成像和结构分析的各种组合。更高的性能，分辨率大大优于1 nm，是可能的，因为目前的结果只受到信噪比的限制。为了实现这一目标，我们在这里申请一种显微镜，它将取代我们建立ONE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提供相同的设置，特别是处理活体样本，以取代我们以前设置的一般功能。简而言之，我们需要一个能够执行以下操作的装置：1）以优异的速度和信噪比提供膨胀凝胶的动态分析，以获得分子级分辨率; 2）允许在超分辨率下对样品进行常规测试。（50 nm或更好）; 3）使样品分析程序能够作为光漂白后的荧光恢复（FRAP）; 4）与能够实现强多路复用的方法兼容，例如通过使用荧光寿命成像。所需的设置将被放置在哥廷根大学医学中心的生物医学显微镜单元，并将取代2007年安装的大量使用的显微镜，该显微镜将在2023年后不再全面运行。