Aberration correction for real-time measurements in adaptive confocal microscopy

生物组织的体内研究的进展基本上依赖于具有不断增加的空间和时间分辨率的显微镜。在本提案中，我们解决这一挑战的目的是提高空间分辨率的共焦激光扫描显微镜使用新的自适应光学元件和控制技术。为了产生三维图像，共焦显微镜用聚焦激光束扫描体积，通常通过移动光学元件并使用压电或振镜，其中速度受到质量和惯性的限制。这可能进一步导致运动伪影并且需要庞大的设置，从而限制了小型化的潜力。光斑大小，因此，分辨率，可以-在原则上-使用具有高数值孔径的显微镜物镜最小化。然而，由于它们是针对具有有限场的单个焦平面进行优化的，因此除了样品引起的像差之外，扫描还导致显著的系统引起的像差，因此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的像差校正和无运动scanning.Our的目标是一个自适应共焦显微镜，理想情况下只使用两个（一个透镜和一个棱镜）自适应光学元件，以提供快速的三维扫描和实时像差校正衍射限制成像在整个视场的发展。为此，我们将开发新型的自适应透镜，结合联合收割机轴向扫描和像差校正，包括几何（散焦，球面，散光，彗差）和色差。同样地，我们将使用新颖的双轴消色差自适应棱镜进行横向扫描。与用振镜扫描相比，这允许更紧凑的共线几何形状。使用这些自适应光学元件，这种新型的智能显微镜具有很大的潜力，小型化和强大的手持系统的发展。我们将通过研究斑马鱼胚胎中致甲状腺肿剂的影响来展示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.