Correlative Refractive Index Light-sheet Microscopy

开发未来疗法是EPSRC的医疗技术大挑战的第一个，对于保持国民健康服务的可持续发展至关重要。目前，估计英国70%的医疗保健支出用于慢性病的管理。再生医学有望显著降低成本，因为它可以将慢性、退行性疾病转变为可治愈的疾病。实现这种潜力需要正确的工具来研究生物发育过程，因为它从单细胞发展到整个器官的复杂结构。光学显微镜的发明，特别是相衬显微镜，使得以前所未有的清晰度突出活细胞的精细特征成为可能。然而，在显微镜载玻片上分离的细胞在其自然的三维环境中往往表现得不像组织。最近发展的平面照明光片显微镜使完整的可视化，荧光标记，生物在发展过程中。虽然光片显微镜对透明的斑马鱼或化学清除的组织非常成功，但许多组织太不透明，无法在第一层细胞之外进行研究。一个典型的例子是处于早期发育阶段的小鸡胚胎。细胞在初始层中复杂的集体行为可以被细致地研究，然而一旦原始条纹形成，在第三维空间中培养胚胎，我们缺乏工具来跟踪细胞的迁移和分化。图像太模糊了。相关折射率光片显微术的目的是使不可见变为可见。它是基于认识到，浊度的生物样品是由于相同的折射率变化产生的结构信息相对比显微镜。样品内光学特性的分布不仅包含有价值的生物学家的结构信息，它也使高分辨率荧光成像所需的自适应波前校正。通过开发一种与荧光图像并行绘制光学性质分布的混合仪器，本提案将实现浑浊生物标本的高分辨率深层组织成像。直接观察生物发育过程的内部运作对于开发有效的再生医学疗法至关重要。

Developing future therapies, the first of EPSRC's Healthcare Technologies Grand Challenges, is essential to keep the National Health Service sustainable. Currently, an estimated 70% of the UK's healthcare expenditure goes towards the management of chronic diseases. Regenerative medicine is expected to significantly reduce costs as it can turn chronic, degenerative, diseases into curable conditions. Realising this potential requires the right tools to study the biological development process as it progresses from the single cell to the complex structure of entire organs.The invention of the optical microscope, and in particular the phase contrast microscope, made it possible to highlight the fine features of living cells with unprecedented clarity. However, cells isolated on a microscope slide often do not behave as tissue in its natural, three-dimensional, environment. The recent development of the planar illumination light-sheet microscope enabled the visualisation of the intact, fluorescently-labelled, organisms during development. While light-sheet microscopy is highly successful for transparent zebrafish or chemically cleared tissue, many tissues are too opaque to be studied beyond the first layer of cells. A prime example is the chick embryo in its early stages of development. The complex collective behaviour of the cells in the initial layer can be studied in exquisite detail, yet as soon as the primitive streak forms, to grow the embryo in the third dimension, we lack the tools to keep track of the cell migration and differentiation. The images are too blurred.Correlative refractive index light-sheet microscopy aims to make the invisible visible. It is based on the realization that the turbidity of biological samples is due to the same refractive index variations that yield structural information in phase-contrast microscopy. The distribution of optical properties within the specimen not only contains valuable structural information for the biologist, it also enables the adaptive wavefront correction needed for high resolution fluorescence imaging. By developing a hybrid instrument that maps the optical property distribution in parallel with the fluorescence image, this proposal will enable high-resolution deep-tissue imaging in turbid biological specimen. A direct view into the inner workings of the biological development process is essential to develop effective regenerative-medicine therapies.