Fourier Phase Contrast Microscopy Technique for Biomedical Research

描述(申请人提供)：相差显微镜是教学和研究实验室中非常有用的设备。这是一个两步过程：(1)分离透过样品的光中的偏离分量和未偏离分量，它们之间具有p/2个相位差；以及(2)获得额外的p/2个相位分离，从而将相位信息转换成用于显示的幅度(强度)对比度。最近，我们展示了一种新的用户友好的傅立叶相差显微镜(FPCM)技术的原理，该技术利用了低功率激光的单色性和相位相干特性以及向列相液晶的光热诱导双折射，具有提高性能的潜力。由于偏移角取决于光源的线宽，因此激光有助于在傅里叶平面上进行分辨率良好的空间频率(对象信息)映射。较高的相位相干性保持了相位延迟。当液晶盒放置在傅立叶平面上时，中心的低空间频率足够强(通过精细控制源强度实现)以诱导局部液晶分子进入各向同性相，而边缘的高空间频率则不那么强烈，并且保持在各向异性液晶相中，从而导致高、低空间频率之间的p/2位相差。因此，它起到了全光自适应可变相位滤光器的作用，并且不需要通过电池两端的电压来对准分子。与商业显微镜获得的图像相比，活体生物标本的初步结果显示出良好的图像对比度(导致显示更多特征以及更清晰地显示相同特征)。由于我们的系统不需要聚光器环-位相板组合(用于商用相差显微镜)，因此图像没有伪影。在R21的应用中，我们将通过优化设计中涉及的各种参数来开发全光自适应FPCM，并与商业仪器相比定量评估该系统的性能，并开发一种用于生物医学研究的新的和改进的傅立叶相差显微镜技术。该系统能够实现多种功能：(1)以改善的图像对比度以微米分辨率成像位相物体；(2)对嵌入在散射介质中的位相物体成像；以及(3)通过在探测器前安装偏振器来区分幅度和位相物体。已经组建了一支由具有物理和生物学专业知识的研究人员组成的多学科团队，以确保彻底调查。我们的主要目标是开发一种坚固、可靠和多功能的系统，不仅可供研究使用，还可用于生物和生物医学实验室的教学。当这项技术的全部潜力被展示时，我们相信这项创新技术提供了几个比目前最先进的仪器设备更好的优势，并可能导致生物医学研究的重大突破。 公共卫生相关性：我们建议优化设计中涉及的各种参数，定量评估系统的性能，并开发用于生物医学研究的傅立叶相差显微镜技术。该系统的创新之处在于利用了相干源、光学傅里叶变换和液晶的光热诱导双折射特性所提供的优势。

DESCRIPTION (provided by applicant): Phase contrast microscope is an extremely useful device in teaching and research labs. It is a two step process: (1) separation of deviated and undeviated components in the light transmitted through the specimen with a p/2 phase difference between them and (2) obtaining an additional p/2 phase separation thereby converting phase information into amplitude (intensity) contrast for display. Recently we demonstrated the principle of a novel user-friendly Fourier phase contrast microscopy (FPCM) technique with potential for enhanced performance exploiting monochromaticity and phase coherence characteristics of a low power laser and photo-thermal induced birefringence of nematic liquid crystal. As the deviation angle depends on the line width of the source, laser facilitates well resolved spatial frequency (object information) mapping in the Fourier plane. High degree of phase coherence preserves the phase retardation. When the liquid crystal cell is placed at the Fourier plane, low spatial frequencies at the center are intense enough (achieved by the fine control of the source intensity) to induce local liquid crystal molecules into isotropic phase whereas high spatial frequencies on the edges are not so intense and remain in the anisotropic liquid crystal phase resulting in p/2 phase difference between high and low spatial frequencies. Thus it acts as an all-optical self-adaptive variable phase filter and no voltage is required across the cell to align the molecules. Preliminary results on live biological specimens show good image contrast (resulting in display of additional features as well as a more clear display of same features) compared to the images obtained with commercial microscopes. Since the condenser annulus - phase plate combination (used in commercial phase contrast microscope) is not required in our system, the images are free from artifacts. In this R21 application, we will develop all-optical self-adaptive FPCM by optimizing various parameters involved in the design, quantitatively asses the performance of the system in comparison to commercial instrumentation and develop a new and improved Fourier phase contrast microscopy technique for biomedical research. This system is capable of performing a variety of functions: (1) imaging phase objects at micrometer resolution with improved image contrast, (2) imaging phase objects embedded in a scattering medium, and (3) discriminating amplitude and phase objects by incorporating a polarizer before the detector. A multidisciplinary team of researchers with expertise in physics and biology has been assembled to ensure thorough investigation. Our main goal is to develop a rugged, reliable and versatile system, readily available not only for research but also teaching in biological and biomedical labs. When full potential of the technique is demonstrated, we believe that this innovative technology offers several advantages over current state of the art instrumentation and may result in significant breakthroughs in biomedical research. PUBLIC HEALTH RELEVANCE: We propose to optimize various parameters involved in the design, quantitatively assess the performance of the system and develop Fourier phase contrast microscopy technique for biomedical research. The novelty of the system lies in exploiting the advantages offered by a coherent source, optical Fourier transformation and photo-thermal induced birefringence property of a liquid crystal.