Aberration correction for real-time measurements in adaptive confocal microscopy

生物组织活体研究的进展基本上依赖于空间和时间分辨率不断提高的显微镜。在目前的方案中，我们旨在通过使用新的自适应光学元件和控制技术来提高共聚焦激光扫描显微镜的空间分辨率来解决这一挑战。为了产生三维图像，共焦显微镜用聚焦的激光束扫描体积，通常是通过移动光学元件和使用速度受质量和惯性限制的压电镜或振镜。这可能会进一步导致运动伪影，并需要庞大的设置，限制了微型化的潜力。原则上，使用具有高数值孔径的显微镜物镜可以最小化光斑大小，从而使分辨率最小化。然而，由于它们是针对有限视场的单个焦平面进行优化的，除了样品诱导的像差之外，扫描还会导致显著的系统诱导像差，从而限制了可实现的分辨率。为了克服这些问题，人们可以使用自适应元件进行像差校正和无运动扫描。我们的目标是开发一种自适应共焦显微镜，理想地只使用两个(一个透镜和一个棱镜)自适应光学元件来提供快速三维扫描和实时像差校正，以便在整个视场内进行衍射限制成像。为此，我们将开发结合轴向扫描和像差校正的新型自适应透镜，包括几何像差(散焦、球面、散光、彗差)和色差。类似地，我们将使用新型的双轴消色差自适应棱镜来预制横向扫描。与振镜扫描相比，这允许更紧凑的共线几何。使用这些自适应光学元件，这种新型的智能显微镜在小型化和开发健壮的手持系统方面具有巨大的潜力。我们将通过研究甲状腺激素对斑马鱼胚胎的影响来展示其潜在的应用和增强的成像性能。

Progress in the in vivo investigation of biological tissue essentially relies on microscopy with ever increasing spatial and temporal resolution. In the present proposal, we address this challenge by aiming to enhance the spatial resolution confocal laser scanning microscopy using novel adaptive optical elements and control techniques. To produce a three dimensional image, confocal microscopy scans the volume with a focused laser beam, typically by moving optical elements and using piezo or galvo mirrors where the speed is limited by mass and inertia. This may further result in motion artefacts and requires a bulky set-up, limiting the potential for miniaturization. The spot size, and hence the resolution, can - in principle - be minimized using microscope objectives with a high numerical aperture. As they are optimized for a single focal plane with a limited field, however, scanning results in significant system-induced aberrations in addition to the sample-induced aberrations and hence limits the achievable resolution. To overcome these problems, one can use adaptive elements both for aberration correction and for motion-free scanning.Our aim is the development of an adaptive confocal microscope that uses ideally only two (one lens and one prism) adaptive optical elements to provide fast three dimensional scanning and real-time aberration correction for diffraction-limited imaging over the whole field of view. For this purpose, we will develop novel adaptive lenses that combine axial scanning and aberration correction, including geometric (defocus, spherical, astigmatism, coma) and chromatic aberrations. Similarly, we will preform the lateral scans with novel bi-axial achromatic adaptive prisms. In contrast to scanning with galvo mirrors, this allows for a more compact collinear geometry. Using these adaptive optical elements, this novel smart microscope has a great potential for miniaturization and for the development of robust handheld systems. We will demonstrate potential applications and the enhanced imaging performance by investigating the effects of goitrogens in zebrafish embryos.