Optical Coherence Tomography for Embryology

拟议的研究计划旨在开发基于光学相干层析成像(OCT)的新的高分辨率胚胎学成像工具，OCT是一种非侵入性成像技术，与共焦显微镜相比，它提供更高的深度分辨率和穿透性，特别是当样品距离显微镜物镜几毫米时。这项活动将由肯特大学坎特伯雷分校物理学院应用光学小组和肯特大学生物科学系细胞与发育生物学小组联合开展。两个团队将合并互补领域的专业知识并汇集资源，组成一个跨学科团队，研究基于OCT的测量和成像平台在特征良好的以及用于胚胎学和细胞成像的新型动物模型上的潜力。这项研究将集中在：1.细胞和胚胎的无标记分析和4D成像的新方法；2.开发同时或连续的OCT/荧光系统，以实现OCT与荧光成像的直接比较，并生成一个能够表达GFP并进行荧光标记的4-D成像平台；3.主要基于蛋白质标记的对比增强程序的开发。这项工作将受益于AOG在开发OCT技术的几个创新方面的先前专业知识，用于眼睛的体内成像和几种类型的组织的体外成像。该活动最初将在AOG内使用全功能OCT系统，该系统是在过去5年在OCT领域进行的积极研究中实施的。开发新的非侵入性成像系统的目的是提供成像细胞和胚胎所需的更高分辨率。因此，一开始，研究将着手于与显微镜相关的改进，如开发专门的高分辨率探头，以响应生物成像的需求。我们将通过成像活的野生型和突变的果蝇胚胎的背部闭合的形态发生过程，首次分析单个Z系列中背部和腹面的细胞和组织运动，来测试这个专门的OCT显微镜系统。此外，研究活动将开发OCT/荧光联合系统，以解决双重目标：(1)与OCT同时或顺序地进行荧光成像，使OCT能够与荧光成像直接比较，并生成一个能够对表达绿色荧光蛋白的组织和胚胎进行4-D成像的平台；(2)开发OCT成像的对比度增强程序，从而能够对细胞结构进行蛋白质标记和深度成像。将设计一个多功能平台来容纳荧光和OCT波段的几种组合，具体配置取决于荧光波段是接近还是叠加到OCT带宽。适应不同频段的OCT操作需要单模光纤传输，这是一项昂贵的工作。因此，我们将设计一种配置，允许在最小的变化下适应最广泛的荧光/OCT波段对。然后，我们将开始评估光敏色素A作为OCT成像的体内造影剂。最近提出的新型非荧光、非生物发光分子成像探针，如泵浦探测OCT、泵浦抑制OCT、基态恢复OCT等，将开创相干光学分子成像的新方向。为相干分子成像设计的探针和技术可能会提高OCT的检测和诊断能力。因此，现在是考虑这种途径的时候了。我们的目标是通过基因工程使果蝇品系在细胞质中表达光敏光敏色素A并将其作为蛋白质标签，从而进一步提高我们在胚胎发育研究中深度进行4-D细胞成像的OCT成像能力。

The proposed research programme aims to develop novel high-resolution imaging tools for Embryology based on optical coherence tomography (OCT), a non-invasive imaging technology that, compared to confocal microscopy, provides enhanced depth resolution and penetration, especially when the sample is several millimetres away from the microscope objective. The activity will be carried jointly, by the Applied Optics Group (AOG) of the School of Physical Sciences and the Cell and Developmental Biology group of the Biosciences Department at the University of Kent at Canterbury. The two teams will amalgamate expertise in complementary areas and pool resources to form an interdisciplinary team to investigate the potential of OCT based measurement and imaging platforms on well-characterised as well as on novel animal models for embryology and cell imaging. The research will focus on: 1. New approaches for label-less analysis and 4D imaging of cells and embryos; 2. Development of a combined simultaneous or sequential OCT/fluorescence system for allowing direct comparison of OCT with fluorescence imaging and generating a platform capable of 4-D imaging GFP-expressing and fluorescently labelled tissues and embryos; 3. Development of contrast enhancement procedures, mainly based on protein-tagging. The work will benefit from prior expertise of the AOG in developing several innovative aspects of the OCT technology for in-vivo imaging of the eye and for in-vitro imaging of several types of tissue. The activity will initially use fully functional OCT systems within the AOG implemented over the last 5 years of active research in the field of OCT. Development of novel non-invasive imaging systems is aimed at providing the much higher resolution required for imaging cells and embryos. Therefore, at the start, the research will embark on microscopy related improvements such as developing specialised high resolution probe heads to respond to the needs of biological imaging. We will test this specialised OCT microscope system by imaging the morphogenetic process of dorsal closure in live wild type and mutant Drosophila embryos, analysing for the first time cell and tissue movements at the dorsal and ventral surfaces in a single Z-series. Further, the research activity will develop a combined OCT/fluorescence system to address a double target: (i) fluorescence imaging simultaneously or sequentially with OCT, allowing direct comparison of OCT with fluorescence imaging and generating a platform capable of 4-D imaging GFP-expressing and fluorescently labelled tissues and embryos, (ii) development of contrast enhancement procedures for OCT imaging, allowing for protein-tagging and deep imaging of cellular structure. A versatile platform will be devised to accommodate several combinations of fluorescence and OCT bands, the exact configuration depending upon whether the fluorescence band is close or superposes to the OCT bandwidth. Accommodating different bands for OCT operation, which requires single mode fibre delivery is an expensive exercise. Therefore, we will devise a configuration that will allow, with minimal changes, adaptation to the widest variety of fluorescence/OCT band pairs. We will then embark on evaluating Phytochrome A as an in vivo contrast agent for OCT imaging. Novel nonfluorescent and nonbioluminescent molecular imaging probes have been proposed recently, such as pump probe OCT, pump suppression OCT, ground state recovery OCT that will initiate new directions in coherent optical molecular imaging. Probes and techniques designed for coherent molecular imaging are likely to improve the detection and diagnostic capabilities of OCT. Therefore it is timely to consider such avenues. We aim to further improve our OCT imaging capability for 4-D cellular imaging at depth in studies of embryonic development by genetically engineering Drosophila strains to express photoactive Phytochrome A cytoplasmically and as a protein tag.