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

细胞运动在哺乳动物胚胎发育过程中发挥着核心作用，提供了决定细胞命运的过程的信息。这一知识不仅对理解发育很重要，而且对人类的治疗也有意义，因为它与胚胎细胞的发展潜力有关，胚胎细胞可能代表干细胞群体。最近培养和成像技术的进步使人们能够观察到这些动态过程，因为它们发生在活的胚胎中。然而，这些方法有几个局限性，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害。这不仅对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结构的变化，需要一种非侵入性的、无标记的成像方法，能够提供高分辨率的四维信息。我们建议在胚胎本身中使用产生二次和三次谐波信号的对比度。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更长波长的光，与其他成像方法(如荧光)相比，光毒性效应显著降低。因此，我们建议开发一种高次谐波(HHG)扫描激光显微镜，用于这一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很简单，但重要的是要记住，产生HHG信号的图像分辨率和效率都严重依赖于扫描样品的焦点的强度和形状。不幸的是，当激发光朝着产生谐波辐射的焦点传播时，它不可避免地穿过具有不同光学性质的样品区域。这会导致像差焦点，从而导致图像信号和分辨率的降低。因此，有必要开发方法来补偿这些不良影响。我们建议使用自适应光学的方法来补偿这些样品引起的像差。该场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。我们的方法特别适用于显微镜而不是天文学，它需要在扫描显微镜的光学系统中使用适当控制的可变形反射镜。该反射镜既用于测量像差的大小，也用于补偿它们，是对HHG显微镜光学系统的唯一改进。通过这种方式，我们将能够消除样品诱导像差的影响，以便在HHG激光扫描显微镜中实现最佳性能。像差矫正的好处之一是能够以较低的激光功率成像发育中的胚胎，从而进一步减少长时间对胚胎发育的影响。这项研究将提供胚胎在最早发育阶段的“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。

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.