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

最近，利用组织类器官细胞培养对复杂细胞-细胞、细胞-基质相互作用进行三维研究的应用重新兴起。这需要新颖的仪器来利用定量功能成像方法，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呈非线性关系。选择性平面成像（SPIM）等方法由于其减少了景深（增加了轴向分辨率）和大视场而优于共聚焦方法。Ameer-Beg小组最近开发了世界上第一个用于成像活斑马鱼功能探针的数字扫描光片荧光寿命成像（FLIM）平台，但该方法存在显着的局限性，包括荧光寿命分辨率和灵敏度。正如我们在共聚焦和多光子成像中所展示的那样，包括大型单光子雪崩光电二极管阵列在内的探测技术的最新进展，在单噪声和动态范围方面提供了潜在的改进。迄今为止，使用光片方法的功能成像是一种未满足的技术需求。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。我们将把这种新的成像平台应用于3D细胞培养和类器官的成像，直接与成熟的2光子薄膜成像进行比较。该学生将根据我们的扫描阵列显微镜专利申请（英国专利申请号1710743.4），研究一种具有荧光寿命成像能力的新型各向同性选择性平面成像技术的发展。使用空间光调制器，我们将在样品中投射一个复杂的三维多光束阵列，该阵列将在正交平面上使用互补重新成像组件进行检测。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，各向同性分辨率，由于增强抑制散射与共聚焦孔径。因此，该系统的独特之处在于其共聚焦孔径排列在Theta显微镜的几何形状中（Stelzer和S. Lindek, Opt. comm . 111, 536-547(1994)）。虽然在双光子激发情况下不需要共聚焦孔径，但在theta显微镜中，单视图激发/检测几何结构通过聚焦激发和孔径检测的组合导致各向同性分辨率。该概念允许扩展到双向（即反向激励/检测）方法，从而进一步提高各向同性分辨率。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双光子面成像模式。新平台的生物学应用可以在3D细胞培养系统和类器官（Maddy Parsons）中设想，以研究细胞底物的相互作用。最初，我们将专注于使用3D打印技术（EnvisionTEC Bioplotter, Ameer-Beg）对具有明确机械性能的工程细胞基质进行成像，以研究众所周知的FRET生物传感器，如Rho GTPase和血管蛋白张力传感器（我们在这方面拥有相当的专业知识），以及专门的EPAC传感器（与阿姆斯特丹Kees Jalink教授合作），以研究响应底物变形和3D分子线索的信号。里程碑：第一年开发双数字扫描光片平台与FLIM检测仪器控制软件开发。知识活动的M平方转移。成像系统在三维类器官中的应用，结合生物绘图和微流体技术。与M Squared合作开发FLIM数据体绘制分析平台。三年级的调查

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