Isotropic 3D Digitally Scanned Light Sheet Time-Domain Fluorescence Lifetime Imaging

各向同性 3D 数字扫描光片时域荧光寿命成像

• 批准号: 2333870
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2333870
• 关键词: Isotropic; 3D; Digitally; Scanned; Light

There has been a recent resurgence in the application of 3-dimensional investigation of complex cell-cell, cell-matrix interactions using tissue organoid cell-culture. This requires novel instrumentation to take advantage of quantitative functional imaging methodologies such as fluorescence resonance energy transfer (FRET) since aberrations and scattering within organoids scale non-linearly with optical pathlength. Methods such as selective plane imaging (SPIM) offer an advantage over confocal methodologies due to their reduced depth of field (increased axial resolution) and large field of view. The Ameer-Beg group recently developed the world's first digitally scanned light sheet fluorescence lifetime imaging (FLIM) platform for imaging functional probes in living Zebrafish but there are significant limitations in the methodology including fluorescence lifetime resolution and sensitivity. Recent advances in detection technology including large arrays of single photon avalanche photodiodes offer potential improvements in single-to-noise and dynamic range as we have shown in both confocal and multiphoton imaging. To date, functional imaging using light sheet methodologies is an unmet technological need. This studentship aims to address this short-fall through development of an instrument which can obtain isotropic resolution over volumes suitable for 3D organotypic cultures. We will apply this novel imaging platform to imaging 3D cell cultures and organoids in direct comparison to well-established 2-photon FLIM. The student will investigate the development of a novel isotropic selective plane imaging technique with fluorescence lifetime imaging capability based on our patent application for Swept array microscopy (UK Patent Application No. 1710743.4). Using a spatial light modulator we will project a complex multibeam array in 3 dimensions into the sample which will be detected in an orthogonal plane using a complementary re-imaging assembly. A cooled SPAD array sensor will be used for detection, acquiring high resolution fluorescence lifetime imaging data with isotropic resolution due to enhanced rejection of scatter with confocal aperturing. As such, this system is unique in having confocal apertures arranged in a Theta microscopy geometry (Stelzer and S. Lindek, Opt. Commun. 111, 536-547 (1994)). Whilst confocal apertures are not required in the 2-photon excitation case, in a theta microscope the single view excitation/detection geometry leads to isotropic resolution through the combination of focused excitation and apertured detection. The concept allows extension to a bidirectional (i.e. reversed excitation/detection) methodology such that isotropic resolution is further improved. The platform offers a unique opportunity to provide interlaced 2-photon en face imaging modes for additional multi-view applications. Biological applications of the new platform can be envisaged in 3D cell culture systems and organoids (Maddy Parsons) to investigate cell substrate interactions. Initially, we will concentrate on imaging of engineered cell matrices with defined mechanical properties using 3D printing technology (EnvisionTEC Bioplotter, Ameer-Beg) to investigate well-understood FRET biosensors such as Rho GTPase and vinculin tension sensors (in which we have considerable expertise), and specialised EPAC sensors (in collaboration with Prof Kees Jalink, Amsterdam) to investigate signalling in response to substrate deformation and molecular cues in 3D. Milestones:Year 1Development of dual digitally scanned light sheet platform with FLIM detectionSoftware development for instrument control. Transfer of knowledge activities with M Squared.Year 2Application of Imaging system to 3D organoids produced using a combination of Bioplotting and microfluidics. Development of analysis platform for volume rendering of FLIM data in collaboration with M Squared.Year 3Investigation

最近，在使用组织类器官细胞培养的复杂细胞-细胞、细胞-基质相互作用的三维研究中的应用已经复苏。这需要新型仪器来利用定量功能成像方法，例如荧光共振能量转移（FRET），因为类器官内的像差和散射与光程长度成非线性比例。诸如选择性平面成像（SPIM）之类的方法由于其减小的景深（增加的轴向分辨率）和大的视场而提供优于共焦方法的优势。Ameer-Beg小组最近开发了世界上第一个数字扫描光片荧光寿命成像（FLIM）平台，用于对活体斑马鱼中的功能探针进行成像，但该方法存在重大局限性，包括荧光寿命分辨率和灵敏度。在检测技术，包括单光子雪崩光电二极管的大型阵列的最新进展提供了潜在的改善单噪声和动态范围，我们已经在共焦和多光子成像。迄今为止，使用光片方法的功能成像是未满足的技术需求。该研究旨在通过开发一种仪器来解决这一不足，该仪器可以在适合3D器官型培养的体积上获得各向同性分辨率。我们将应用这种新型成像平台对3D细胞培养物和类器官进行成像，与成熟的2光子FLIM进行直接比较。学生将研究一种新的各向同性选择性平面成像技术的发展与荧光寿命成像能力的基础上，我们的专利申请扫描阵列显微镜（英国专利申请号1710743.4）。使用空间光调制器，我们将在3维中投射复杂的多光束阵列到样品中，该样品将使用互补的再成像组件在正交平面中被检测。一个冷却的SPAD阵列传感器将用于检测，获得高分辨率的荧光寿命成像数据与各向同性的分辨率，由于增强拒绝与共焦孔径的散射。因此，该系统的独特之处在于具有以Theta显微镜几何形状布置的共焦孔径（Stelzer和S. Lindek，Opt. Commun. 111，536-547（1994））。虽然共焦孔径是不需要在2光子激发的情况下，在θ显微镜的单视图激发/检测几何形状导致各向同性的分辨率通过组合的聚焦激发和孔径检测。该概念允许扩展到双向（即反向激发/检测）方法，使得各向同性分辨率进一步提高。该平台提供了一个独特的机会，为额外的多视图应用提供交错的2光子正面成像模式。新平台的生物学应用可以设想在3D细胞培养系统和类器官（Maddy Parsons）中研究细胞基质相互作用。最初，我们将专注于使用3D打印技术对具有确定机械性能的工程细胞基质进行成像（EnvisionTEC Biotechnology，Ameer-Beg）以研究熟知的FRET生物传感器，例如Rho GTdR和黏着斑蛋白张力传感器（我们在这方面拥有相当的专业知识），以及专门的EPAC传感器（与Kees Jalink教授合作，阿姆斯特丹）研究3D中响应基底变形和分子线索的信号传导。里程碑：Year 1开发具有FLIM检测功能的双数字扫描光片平台仪器控制软件开发。与M Squared一起进行知识传授活动。第2年将成像系统应用于使用生物绘图和微流体技术相结合产生的3D类器官。与M Squared合作开发FLIM数据体绘制分析平台。