DIGITAL OPTICAL PHASE CONJUGATION ASSISTED 4Pi FLUORESCENCE MICROSCOPY

描述(由申请人提供)：这项探索性/发展研究(R21)申请建议开发一种光学相位共轭辅助(OPC)4PI显微镜，能够进行高横向(200 Nm)和轴向(120 Nm)分辨率成像。典型的4PI显微镜由聚焦到同一空间点的两束反向传播的激光组成。两束光束的相互干涉使轴向分辨率比标准共焦显微镜提高了5-7倍。然而，4PI显微镜对光学不均匀样品的性能很差，因为广泛的散射会降低焦点质量，并可能导致两个焦点的不对准。此外，4PI显微镜通常很难实现和很好地使用，因为激光光束焦点的准确叠加非常关键，但很容易受到干扰。我们提议的研究将利用我们目前在光学相位共轭(OPC)、通过OPC抑制时间反转浑浊以及组织光学方面的专业知识来显著提高4PI显微镜的能力。光学相位共轭可以粗略地解释为目标光场的时间反转回放。我们最近证实，因为散射是确定性的，所以有可能通过时间反转穿过目标组织的散射光场来撤销组织散射。我们的目的是证明，在4PI显微镜中加入特殊调整的OPC装置将使这种显微镜能够自适应地对准其焦距并校正样品像差，这一直是传统4PI系统的障碍。提出的OPC 4PI显微镜具有广阔的应用前景。预期的像差补偿能力将允许该系统成像更厚和更不均匀的样品。我们预计，这种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)的精确3D映射非常重要。 与公共卫生相关：这一拟议项目的成功完成将带来更强大的4PI显微镜系统，这些系统更易于使用，并且能够通过更厚和更不均匀的样本进行成像。我们预计，这种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)的精确3D映射非常重要。这项拟议的工作还将进一步推动我们目前在使用时间反转光场诱导组织透明方面的研究工作-这一研究方向最终可以导致准确和非侵入性的生化传感，以及基于光的深层组织成像。

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.