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起着核心作用，提供了有关决定细胞命运的过程的信息。这一知识不仅对了解发育很重要，而且对人类治疗也有影响，因为它涉及可能代表干细胞群体的胚胎细胞的发育潜力。培养和成像技术的最新进展允许观察这些动态过程，因为它们发生在活胚胎中。然而，这些方法受到几个限制，主要的一个是它们依赖于染料或蛋白质的荧光来可视化细胞，由于组织吸收光能和加热，导致对胚胎的显著累积损伤。这不仅对所获取的图像数据的分辨率施加了限制，而且还可能干扰培养胚胎的发育。为了观察发育中胚胎的结构变化，需要一种非侵入性、无标记的成像方法，可以提供高分辨率的四维信息。我们建议使用来自胚胎本身的二次和三次谐波信号的产生的对比度。这些信号的产生不需要使用任何外源性标记，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，导致光毒性效应显著降低。因此，我们建议开发一个高次谐波产生（HHG）扫描激光显微镜，这个生物学上的重要和具有挑战性的应用。虽然这样的显微镜在概念上是简单的，重要的是要记住，图像分辨率和HHG信号产生的效率都严重依赖于焦斑的强度和形状，它扫描整个标本。不幸的是，这是不可避免的，因为激发光传播的焦点，在那里产生的谐波辐射，它通过具有不同的光学特性的样品的区域。这导致像差焦斑，其导致图像信号和分辨率两者的降低。因此，有必要开发补偿这些不良影响的方法。我们建议采用自适应光学的方法来补偿这些标本引起的像差。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事望远镜的图像而开发的。我们的方法，这是特别适合于显微镜，而不是天文学，需要使用一个适当控制的变形镜的光学系统的扫描显微镜。反射镜用于测量像差的大小并对其进行补偿，是对HHG显微镜光学系统的唯一修改。通过这种方式，我们将能够消除样品引起的像差的影响，从而允许在高分子量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，从而进一步减少对胚胎长期发育的影响。这项研究将提供胚胎发育早期阶段的“地图集”，包括细胞位置和运动的三维地图。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