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，只能达到成像光波长的大约一半（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屏障，但荧光显微镜。仍然无法对单个蛋白质或小分子复合物的形态进行成像，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，其中样品在嵌入后被放大。原则上，可膨胀凝胶应该达到分子分辨率，但这种方法失败了，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）。我们最近找到了解决这个问题的方法，采用第三类光学超分辨率方法，该方法基于对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相结合。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，从获得的诊断到单个形状的分析。一种显微镜为生物科学开辟了许多新途径，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，再到更高的性能。优于 1 nm 是可能的，因为当前结果仅受信噪比的限制。为了实现这一目标，我们在此申请了一种显微镜，它将取代我们建立 ONE 显微镜的装置，同时提供显着的优势。此外，我们需要对许多其他项目进行相同的设置，特别是处理活样本，以替换我们之前设置的一般功能。简而言之，我们需要一个能够执行以下操作的设置：1）提供动态分析。膨胀的凝胶具有出色的速度和信噪比、分子尺度分辨率；2) 允许在固定细胞和活细胞中以超分辨率（50 nm 或更高）对样品进行常规测试；3) 使样品分析程序能够在荧光恢复后进行光漂白（FRAP）；4）与能够实现强多重分析的方法兼容，例如使用荧光寿命成像，所需的装置将放置在哥廷根大学医学中心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​​，2023 年后将不再全面运行。