Phillips Kevin G, Baker-Groberg Sandra M, McCarty Owen J T
Department of Biomedical Engineering, Oregon Health & Science University, School of Medicine; Department of Dermatology, Oregon Health & Science University, School of Medicine;
Department of Biomedical Engineering, Oregon Health & Science University, School of Medicine.
J Vis Exp. 2014 Apr 7(86):50988. doi: 10.3791/50988.
We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using "off-the-shelf" microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.
我们描述了如何使用标准光学显微镜，通过明场和微分干涉对比成像相结合的方式，对细胞标本的质量、体积和密度进行定量测量。本文介绍了两种主要方法：非干涉定量相显微镜（NIQPM），用于测量细胞总质量和亚细胞密度分布；希尔伯特变换微分干涉对比显微镜（HTDIC），用于确定体积。NIQPM基于一种简化的波传播模型，即傍轴近似，有三个基本假设：低数值孔径（NA）照明、弱散射和标本对光的弱吸收。幸运的是，未染色的细胞标本满足这些假设，并且在商业显微镜上很容易实现低NA照明。HTDIC用于在高NA照明条件下从聚焦扫描DIC图像中获取体积信息。高NA照明能够增强标本沿光轴的切片效果。对DIC图像堆栈进行希尔伯特变换处理，通过将标本强度的灰度值与背景的灰度值分离，极大地增强了用于在三维空间中定位标本边界的边缘检测算法。NIQPM和HTDIC的主要优点在于它们可以使用“现成的”显微镜进行技术操作。这些方法有两个基本局限性：目前商业显微镜上的z轴堆栈采集时间较慢，无法对速度超过每分钟1帧的现象进行研究；其次，衍射效应将NIQPM和HTDIC的应用分别限制在直径为0.2至10μm（NIQPM）和20μm（HTDIC）的物体上。因此，感兴趣的标本及其相关的时间动态必须满足一定的尺寸和时间限制，才能使用这些方法。令人兴奋的是，大多数固定的细胞标本都可以用这些方法轻松研究。
