Unlocking the potential of single-photon wide-field microscopy

释放单光子宽视场显微镜的潜力

• 批准号: EP/Y022491/1
• 负责人: Dirk-Peter Herten
• 金额: $ 80.55万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FY022491%2F1
• 关键词: Unlocking; potential; single; photon; wide

This project aims at transforming wide-field microscopy by adding single photon capabilities that are so far only available in confocal microscopy. We will use the latest generation of single photon sensitive imaging sensors with improved photon detection efficiency and embedded data processing circuits to enable the recording of single photons at MHz rate with picosecond accuracy. This will significantly reduce the readout noise and enable different new imaging modes in wide-field microscopy such as fluorescence lifetime imaging and photon correlation imaging which can be used for multiplexing or molecular counting. In this project, we will focus on improving different super-resolution microscopy techniques for imaging fixed and living cells. For instance, the localisation precision in single-molecule localisation microscopy (SMLM) will benefit not only from the reduced noise levels but also from the possibility to distinguish and reject instantly scattered light from the delayed light emission of the fluorescent labels. Moreover, the embedded photon histogramming capabilities will allow to implement fluorescence lifetime-based multiplexing and thereby increase the number of different labels that can be used in a sample. We will also explore the potential for probing molecular interactions in cells using single-molecule Forster Resonance Energy Transfer. Overall, we expect that this will improve the achievable resolution, add inherent quantitative information to the image data and increase the number of proteins that can be simultaneously imaged. We will explore the application of single-photon SMLM by imaging protein complexes of tetraspanins in the plasma membrane of fixed cells.Furthermore, we want to increase the spatial resolution of super-resolution optical fluctuation imaging (SOFI) and enhance its capabilities in live-cell super-resolution microscopy. Here, the high time resolution of the single photon sensitive imaging sensor will allow us to extend the timescale that can be used for correlating the intensity fluctuations in SOFI. This is very relevant because the majority of optical fluctuations occur on the microsecond timescale which is not covered by current imaging sensors such as EMCCD and sCMOS cameras. Thereby, we expect a significant improvement in the achievable resolution and at the same time a better separation of fluorophores with different properties. We also expect to overcome a major limitation in SOFI by increasing the number of fluorescent labels that can be used in SOFI experiments. Like for single-photon SMLM, we will implement time gating and fluorescence lifetime multiplexing to further reduce noise and increase the number of probes that can be simultaneously imaged. We will explore the suitability of single-photon SOFI by imaging visualising the same protein complexes of tetraspanins and their dynamics in the plasma membrane of living cells.Overall, we expect that the single-photon super-resolution microscopy developed in this project will significantly improve the achievable spatial resolution due to a significantly reduced noise level and rejection of immediate scattering. At the same time, single-photon wide-field microscopy will enable additional imaging modes such as fluorescence lifetime imaging microscopy (FLIM) which will increase the number of simultaneously imaged targets, and photon correlation imaging which will enable quantitative molecular imaging as the first quantum imaging technique in wide-field fluorescence microscopy. The successful development of single-photon super-resolution microscopy will be door opener for other imaging modes such as fluorescence correlation imaging of fast molecular processes. In summary, this will lead to a step change in wide-field microscopy with great prospect to transform the way we can image molecular scale structures and processes in the life sciences and in biomedical research.

该项目旨在通过添加到目前为止仅在共聚焦显微镜中可用的单个光子功能来改变宽视野显微镜。我们将使用具有提高的光子检测效率和嵌入数据处理电路的最新一代单光子敏感成像传感器，以凭借精确度以MHz速率记录单光子。这将显着降低读取噪声，并在宽视野显微镜中启用不同的新成像模式，例如荧光寿命成像和光子相关成像，可用于多重或分子计数。在这个项目中，我们将专注于改进用于成像固定和活细胞成像的不同超分辨率显微镜技术。例如，单分子定位显微镜（SMLM）中的定位精度不仅将从降低的噪声水平中受益，而且还可以从荧光标签的延迟光发射中分开和拒绝立即散射光线的可能性。此外，嵌入式光子直方图功能将允许实现基于荧光寿命的多路复用，从而增加了可以在样品中使用的不同标签的数量。我们还将探索使用单分子Forster共振能传递探测细胞中分子相互作用的潜力。总体而言，我们预计这将改善可实现的分辨率，在图像数据中添加固有的定量信息，并增加可以同时成像的蛋白质数量。我们将通过对固定细胞质膜中四叠蛋白的蛋白质复合物进行成像的蛋白质复合物的成像探索。Furthermore，我们希望增加对超分辨率光波动成像（SOFI）的空间分辨率，并增强其在实用电池超级分辨率显微镜中的能力。在这里，单个光子敏感成像传感器的高时间分辨率将使我们能够延长可用于关联SOFI中强度波动的时间表。这是非常相关的，因为大多数光波动发生在微秒的时间尺度上，该时间表不受当前成像传感器（例如EMCCD和SCMOS摄像机）的覆盖。因此，我们期望可实现的分辨率有显着改善，同时又可以更好地分离具有不同特性的荧光团。我们还期望通过增加可用于SOFI实验的荧光标签的数量来克服SOFI的主要局限性。与单光子SMLM一样，我们将实施时间门控和荧光寿命多路复用，以进一步降低噪声并增加可以同时成像的探针数量。我们将通过成像可视化四叠蛋白酶的相同蛋白质复合物及其在活细胞的质膜中的动态来探索单光沙发的适用性。在这个项目中，我们预计，该项目在该项目中开发的单光子超分辨率显微镜将显着改善由于可获得的空间分辨率可显着改善由于即时降低噪声水平而显着降低噪声水平。同时，单光子宽视野显微镜将启用其他成像模式，例如荧光寿命成像显微镜（FLIM），这些成像显微镜（FLIM）将增加同时成像靶标的数量以及光子相关成像，从而将定量分子成像作为宽阔场中的第一个量子成像技术在宽面积荧光显微镜中作为第一个量子成像技术。单光子超分辨率显微镜的成功开发将是其他成像模式的开门机，例如快速分子过程的荧光相关成像。总而言之，这将导致宽视野显微镜的逐步变化，前景很大，可以改变生命科学和生物医学研究中的分子尺度结构和过程的方式。