Aberration correction for real-time measurements in adaptive confocal microscopy

生物组织体内研究的进展主要依赖于显微镜，其空间和时间分辨率不断提高。在目前的建议中，我们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，共聚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或galvo反射镜。这可能会进一步导致运动伪影，并且需要一个庞大的设置，限制了小型化的潜力。光斑大小，因而分辨率，可以-原则上-最小化使用显微镜物镜与高数值孔径。然而，由于它们是针对具有有限视场的单焦平面进行优化的，除了样品引起的像差外，扫描还会导致显着的系统引起的像差，从而限制了可实现的分辨率。为了克服这些问题，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聚焦显微镜，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，为整个视场的衍射限制成像提供快速三维扫描和实时像差校正。为此，我们将开发新型自适应透镜，结合轴向扫描和像差校正，包括几何（离焦，球面，散光，彗差）和色差。同样，我们将使用新型双轴消色差自适应棱镜进行横向扫描。与galvo反射镜扫描相比，这允许更紧凑的共线几何。使用这些自适应光学元件，这种新型智能显微镜在小型化和开发强大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在斑马鱼胚胎中的作用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.