DIGITAL OPTICAL PHASE CONJUGATION ASSISTED 4Pi FLUORESCENCE MICROSCOPY

描述（由申请人提供）：本探索性/发展性研究（R21）申请建议开发一种光学相位共轭辅助（OPC） 4倍显微镜，该显微镜能够进行高横向（200纳米）和轴向（120纳米）分辨率成像。一个典型的4倍显微镜由两个反向传播的激光束组成，聚焦在同一个空间点上。两束光束的相互干涉允许在轴向分辨率的一个因素5-7相对于标准共聚焦显微镜的改进。然而，对于光学不均匀的样品，4Pi显微镜表现不佳，因为广泛的散射降低了焦点光斑的质量，并可能导致两个焦点光斑的错位。此外，4倍π显微镜通常难以实现和使用，因为激光束焦点的精确叠加是至关重要的，但很容易被破坏。我们提出的研究将利用我们目前在光学相位共轭（OPC）、通过OPC抑制时间反转浊度和组织光学方面的专业知识，显著提高4Pi显微镜的能力。光学相位共轭可以大致解释为目标光场的时间反转回放。我们最近已经确定，由于散射是确定性的，有可能通过时间逆转组织散射-通过目标组织的散射光场。我们的目标是证明在4Pi显微镜中加入特别适应的OPC安排将允许这样的显微镜自适应地对准焦点并纠正样品像差，这一直是传统4Pi系统的障碍。所提出的OPC 4Pi显微镜的应用范围是潜在的广泛。预期的像差补偿能力将允许该系统成像厚和更多的异质样品。我们期望这样的系统将在神经科学和发育生物学实验中有用，在这些实验中，对复杂样本（如胚胎）进行精确的3D绘图是很重要的。

DESCRIPTION (provided by applicant): This Exploratory/Developmental Research (R21) application proposes to develop an optical-phase- conjugation-assisted (OPC) 4Pi microscope that is capable of high lateral (200 nm) and axial (120 nm) resolution imaging. A typical 4Pi microscope consists of two counter-propagating laser beams that are focused to the same spatial point. The mutual interference of the two beams allows an improvement in the axial resolution by a factor of 5-7 versus a standard confocal microscope. However, a 4Pi microscope performs poorly for optically inhomogeneous samples as extensive scattering degrades the focal spot quality and can lead to a misalignment of the two focal spots. Furthermore, 4Pi microscopes are generally difficult to implement and use well because the exact superposition of the laser beams' focal spots is critical but easily disrupted. Our proposed research will leverage our current expertise in optical phase conjugation (OPC), time-reversal turbidity suppression via OPC, and tissue optics to significantly improve the capability of 4Pi microscopy. Optical phase conjugation can be roughly interpreted as the time-reversed playback of a target light field. We have recently established that, because scattering is deterministic, it is possible undo tissue scattering by time- reversing a scattered light field through the target tissue. We aim to demonstrate that the incorporation of a specially adapted OPC arrangement in a 4Pi microscope will allow such a microscope to adaptively align its focus and to correct sample aberration which has been an obstacle for the conventional 4Pi system. The application range of the proposed OPC 4Pi microscope is potentially broad. The anticipated aberration compensation ability will allow this system to image thicker and more heterogeneous samples. We expect that such systems will be useful in neuroscience and developmental biology experiments where precise 3D mapping of complex samples, such as embryos, are important. PUBLIC HEALTH RELEVANCE: The successful completion of this proposed project will result in more robust 4Pi microscope systems that are simpler to use and capable of imaging through thicker and more heterogeneous samples. We expect that such systems will be useful in neuroscience and developmental biology experiments where precise 3D mapping of complex samples, such as embryos, are important. The proposed work will also further our current research effort on the use of time-reversed light field to induce tissue transparency - a research direction that can ultimately lead to accurate and non-invasive biochemical sensing, and light-based deep tissue imaging.