High Resolution 3D Optics Coherence Phase Microscopy

描述（由申请人提供）：细胞和亚细胞结构的三维成像在细胞生理学和医学研究中具有巨大的潜力，因为它可以观察各种环境下的细胞行为。迄今为止，许多三维成像技术都需要对细胞进行染色，以产生通常无法观察到的结构图像。我们提出了一种基于光谱域光学相干断层扫描（SD-OCT）技术的三维光学相干相对比显微镜（OCPM），该显微镜通过测量高灵敏度的深度分辨相位差来揭示细胞在自然状态下的三维结构和动力学。OCPM将与双光子显微镜（TPM）集成为标准倒置显微镜。相位灵敏度、横向分辨率和结构成像能力将在校准对象和细胞上进行评估，其中纳米轴向和亚微米横向分辨率是目标。OCPM图像的解释将通过同一标本的同时双光子荧光成像进行研究。OCPM的潜力将通过成像细胞对外部刺激的反应来研究。深层组织成像（250¿m）的能力和相位灵敏度的利用将通过监测嵌入较大神经束的轴突的动作电位来研究。神经活动已被证明可以诱导1-3纳米量级的神经快速变化（肿胀）。OCPM的时空分辨率能够检测到动作电位传播过程中的这些快速变化。OCPM将作为细胞生物学过程的强大成像工具，并将增强对活细胞结构，功能和行为的理解。本研究的总体目标是：A)开发一种用于细胞三维高分辨率相位对比成像的OCPM系统；B)确定OCPM的相位分辨率和切片能力；C)研究OCPM在外部刺激下细胞动力学研究中的潜力；D)无创测量神经束内轴突的动作电位。

DESCRIPTION (provided by applicant): Three-dimensional imaging of cells and sub cellular structures has enormous potential in studies on cell physiology and medicine, since it enables the observation of cell behavior in a variety of environments. Many three-dimensional imaging techniques to date require cells to be stained or dyed to produce images of structures that are normally not observable. We propose to develop a three-dimensional optical coherence phase-contrast microscope (OCPM) based on Spectral Domain Optical Coherence Tomography (SD-OCT) technology that can reveal three-dimensional structures and dynamics of cells in their natural state by measuring depth resolved phase differences with high sensitivity. The OCPM will be integrated with Two Photon Microscopy (TPM) into a standard inverted microscope. Phase sensitivity, lateral resolution, and structural imaging capability will be evaluated on calibration objects and cells, where nanometer axial and sub-micron lateral resolution are targeted. Interpretation of OCPM images will be investigated by simultaneous two-photon fluorescence imaging of the same specimen. The potential of OCPM will be studied by imaging cell responses to external stimuli. The ability for deep tissue imaging (250 ¿m) and the exploitation of phase sensitivity will be investigated by monitoring action potentials in axons embedded in larger nerve bundles. Neural activity has been demonstrated to induce rapid changes in nerves (swelling) on the order of 1-3 nanometers. The spatio-temporal resolution of OCPM is capable of detecting these rapid changes during action potential propagation. OCPM will serve as a powerful imaging tool for processes in cell biology and will enhance the understanding of the structure, function, and the behavior of living cells. The overall goal of this research is A) to develop an OCPM system for 3 dimensional high resolution phase contrast imaging of cells, B) to determine the phase resolution and sectioning capability of OCPM, C) to investigate the potential of OCPM for the study of cell dynamics in response to external stimuli, D) to non- invasively measure action potentials of axons within nerve bundles. PUBLIC HEALTH RELEVANCE: Three-dimensional imaging of cells and sub cellular structures has enormous potential in studies on cell physiology and medicine, since it enables the observation of cell behavior in a variety of environments. Many three-dimensional imaging techniques to date require cells to be stained or dyed to produce images of structures that are normally not observable. We will develop a new form of microscopy (OCPM) that is able to detect minute changes in cells on the order of nanometers. OCPM will serve as a powerful imaging tool for processes in cell biology and will enhance the understanding of the structure, function, and the behavior of living cells.