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

最近，在利用组织器官细胞培养对复杂的细胞-细胞、细胞-基质相互作用的三维研究中，出现了一种复兴的应用。这需要新的仪器来利用定量功能成像方法，如荧光共振能量转移(FRET)，因为有机体内的像差和散射与光程长度呈非线性关系。像选择平面成像(SPIM)这样的方法比共聚焦方法更具优势，因为它们的景深减小(轴向分辨率增加)，视场更大。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，用于对活斑马鱼的功能探针进行成像，但该方法在荧光寿命分辨率和灵敏度方面存在重大限制。探测技术的最新进展，包括大规模的单光子雪崩光电二极管阵列，提供了潜在的单噪声比和动态范围的改进，正如我们在共焦和多光子成像中所显示的那样。到目前为止，使用光片方法的功能成像是一项尚未得到满足的技术需求。这个项目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。我们将把这个新的成像平台应用于3D细胞培养和有机化合物的成像，直接与公认的双光子薄膜进行比较。学生将基于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开发。利用空间光调制器，我们将把三维复杂的多光束阵列投射到样品中，该样品将使用互补的再成像组件在正交平面上进行检测。将使用冷却的SPAD阵列传感器进行探测，由于共焦孔径增强了对散射的抑制，因此可以获得各向同性分辨率的高分辨率荧光寿命成像数据。因此，该系统的独特之处在于共焦孔以Theta显微镜几何形状排列(Stelzer和S.Lindek，Opt。交警。111,536-547(1994))。虽然在双光子激发的情况下不需要共焦光圈，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有光圈检测的组合导致各向同性分辨率。该概念允许扩展到双向(即反向激发/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，可以为其他多视角应用提供隔行扫描的双光子面内成像模式。新平台的生物学应用可以设想在3D细胞培养系统和有机化合物(Maddy Parsons)中，以研究细胞底物的相互作用。首先，我们将专注于使用3D打印技术(EnvisionTEC BioPlotter，AMEER-BEG)对具有定义机械特性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vinculin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(与阿姆斯特丹的Kees Jalink教授合作)，以研究3D中响应底物变形的信号和分子线索。里程碑：第1年开发具有薄膜探测功能的双数字扫描光片平台用于仪器控制的软件开发。利用M平方图传递知识活动。2年2影像系统在生物标绘和微流控相结合生产的3D有机物中的应用。与M Square合作开发电影数据体绘制分析平台

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