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

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.

Cell movements play a central role throughout mammalian embryogenesis, for example during gastrulation, the translocation of germ cells into the gonads and the formation of neural crest derived structures. An understanding of such dynamic processes is important in integrating our knowledge of molecular and genetic regulatory networks into the context of cellular interactions during embryogenesis. The movement of cells during development is also intimately connected to their ultimate fate. Recent advances in culture and imaging techniques have opened up the possibility of studying these dynamic processes as they happen in living embryos, but these approaches suffer from several limitations, most particularly the use of fluorescence microscopy, resulting in significant cumulative photo-damage to the embryo. This imposes a limit on the spatial and temporal resolution of the image data and is also likely to perturb the development of the cultured embryo. To minimise phototoxicity induced during imaging, we will build a harmonic generation microscope, taking advantage of the non-linear processes of second and third harmonic generation, to perform long term 3D imaging of developing mouse embryos. Significant but correctable specimen-induced optical aberrations are introduced when focussing through such embryos. The consequent reduction in focal spot intensity has a compound effect on the non-linear harmonic generation process. We will therefore use adaptive optics to compensate the aberrations, restoring resolution and contrast. This will in turn permit the use of lower illumination powers, reduced phototoxic effects, increased embryo viability and longer observation times. We will observe embryo development over periods of hours to days and construct a four dimensional model of early embryonic development that can be interrogated for the fates of individual cells. This information will be made publicly available to others in the research community.