Adaptive harmonic generation microscopy: non-invasive imaging for early embryogenesis

自适应谐波发生显微镜：早期胚胎发生的非侵入性成像

• 批准号: BB/E004946/1
• 负责人: Tony Wilson
• 金额: $ 85.99万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE004946%2F1
• 关键词: Adaptive; harmonic; generation; microscopy; non

Cell movements play a central role throughout mammalian embryo development, providing information about the processes that determine cell fate. This knowledge is important not only for an understanding of development, but also has implications for therapy in humans, as it relates to the developmental potential of embryonic cells that may represent populations of stem cells. Recent advances in culture and imaging techniques have permitted observation of these dynamic processes as they happen in living embryos. However, these approaches suffer from several limitations, the major one being that they rely on the fluorescence of dyes or proteins to visualise cells, resulting in significant cumulative damage to the embryo as a result of the tissues absorbing light energy and heating up. This not only imposes a limit on the resolution of the image data acquired, but also is likely to perturb the development of the cultured embryo. In order to observe the changing structure of developing embryos, a non-invasive, label free imaging method that can provide high resolution, four-dimensional information is required. We propose to use the contrast derived from the generation of second and third harmonic signals in the embryos themselves. The generation of these signals does not require the use of any exogenous labels and the need to use longer wavelength light, which is not absorbed by the embryo, results in a significant reduction of phototoxic effects compared to other imaging methods, such as fluorescence. We therefore propose to develop a higher harmonic generation (HHG) scanning laser microscope for this biologically important and challenging application. Although such a microscope is conceptually straightforward, it is important to remember that both the image resolution and the efficiency with which the HHG signal is generated depend critically on the intensity and shape of the focal spot, which scans across the specimen. Unfortunately, it is inevitable that as the excitation light propagates towards the focus, where the harmonic radiation is generated, it passes through regions of the specimen having different optical properties. This results in an aberrated focal spot that leads to a reduction in both image signal and resolution. It is therefore necessary to develop methods to compensate for these undesirable effects. We propose to employ methods of adaptive optics to compensate for these specimen induced aberrations. The field was originally developed for improving images from astronomical and military telescopes by compensating for the effects of the turbulent atmosphere. Our approach, which is particularly appropriate for microscopy rather than astronomy, requires the use of a suitably controlled deformable mirror in the optical system of the scanning microscope. The mirror, which is used both to measure the size of the aberrations as well as to compensate for them, is the only modification to the optical system of the HHG microscope. In this way, we will be able to remove the effects of specimen induced aberrations so as to permit optimal performance in HHG laser scanning microscopes. Among the benefits of aberration correction is the ability to image developing embryos with lower laser powers, thus reducing further the effects on the embryo development over long periods of time. The research will provide an 'atlas' of the development of embryos over the earliest stages of development, including three-dimensional maps of cell locations and movements. This data will be made freely available for the use of other researchers.

细胞运动在整个哺乳动物胚胎发育过程中发挥着核心作用，提供有关决定细胞命运的过程的信息。这些知识不仅对于理解发育很重要，而且对人类治疗也具有影响，因为它与可能代表干细胞群的胚胎细胞的发育潜力有关。培养和成像技术的最新进展允许观察活胚胎中发生的这些动态过程。然而，这些方法存在一些局限性，其中主要的一个是它们依赖染料或蛋白质的荧光来可视化细胞，由于组织吸收光能和升温，导致对胚胎造成显着的累积损伤。这不仅限制了所获取图像数据的分辨率，而且还可能干扰培养胚胎的发育。为了观察发育中胚胎结构的变化，需要一种能够提供高分辨率、四维信息的非侵入性、无标记成像方法。我们建议使用胚胎本身产生的二次和三次谐波信号所产生的对比度。这些信号的产生不需要使用任何外源标记，并且需要使用不被胚胎吸收的较长波长的光，与其他成像方法（例如荧光）相比，光毒性效应显着减少。因此，我们建议为这一具有重要生物学意义且具有挑战性的应用开发高次谐波发生（HHG）扫描激光显微镜。尽管这种显微镜在概念上很简单，但重要的是要记住，图像分辨率和 HHG 信号生成的效率在很大程度上取决于扫描样本的焦点的强度和形状。不幸的是，当激发光向产生谐波辐射的焦点传播时，它不可避免地会穿过样本中具有不同光学特性的区域。这会产生像差焦点，从而导致图像信号和分辨率降低。因此，有必要开发方法来补偿这些不良影响。我们建议采用自适应光学方法来补偿这些样本引起的像差。该领域最初是为了通过补偿湍流大气的影响来改善天文和军用望远镜的图像而开发的。我们的方法特别适合显微镜而不是天文学，需要在扫描显微镜的光学系统中使用适当控制的可变形镜。该镜子既用于测量像差的大小，也用于补偿像差，是对 HHG 显微镜光学系统的唯一修改。通过这种方式，我们将能够消除样本引起的像差的影响，从而实现 HHG 激光扫描显微镜的最佳性能。像差校正的好处之一是能够用较低的激光功率对发育中的胚胎进行成像，从而进一步减少长期对胚胎发育的影响。该研究将提供胚胎发育最早阶段的“图谱”，包括细胞位置和运动的三维图。这些数据将免费提供给其他研究人员使用。

项目成果

Adaptive harmonic generation microscopy of mammalian embryos.

• DOI: 10.1364/ol.34.003154
• 发表时间: 2009-10-15
• 期刊: Optics letters
• 影响因子: 3.6
• 作者: Jesacher A;Thayil A;Grieve K;Débarre D;Watanabe T;Wilson T;Srinivas S;Booth M
• 通讯作者: Booth M

Towards understanding the roles of position and geometry on cell fate decisions during preimplantation development.

• DOI: 10.1016/j.semcdb.2015.09.006
• 发表时间: 2015-12
• 期刊: Seminars in cell & developmental biology
• 影响因子: 7.3
• 作者: Biggins JS;Royer C;Watanabe T;Srinivas S
• 通讯作者: Srinivas S

Image-based adaptive optics for two-photon microscopy.

• DOI: 10.1364/ol.34.002495
• 发表时间: 2009-08-15
• 期刊: Optics letters
• 影响因子: 3.6
• 作者: Débarre D;Botcherby EJ;Watanabe T;Srinivas S;Booth MJ;Wilson T
• 通讯作者: Wilson T

The influence of aberrations in third harmonic generation microscopy

• DOI: 10.1088/2040-8978/12/8/084009
• 发表时间: 2010-08
• 期刊: Journal of Optics
• 影响因子: 2.1
• 作者: A. Thayil;A. Jesacher;T. Wilson;M. Booth

Use of the viral 2A peptide for bicistronic expression in transgenic mice.

• DOI: 10.1186/1741-7007-6-40
• 发表时间: 2008-09-15
• 期刊: BMC BIOLOGY
• 影响因子: 5.4
• 作者: Trichas, Georgios;Begbie, Jo;Srinivas, Shankar
• 通讯作者: Srinivas, Shankar