Errington R J, Fricker M D, Wood J L, Hall A C, White N S
Department of Physiology, University of Oxford, United Kingdom.
Am J Physiol. 1997 Mar;272(3 Pt 1):C1040-51. doi: 10.1152/ajpcell.1997.272.3.C1040.
Regulation of cell volume is a fundamental cellular homeostatic mechanism in the face of osmotic stress. In normal articular cartilage, chondrocytes are exposed to a changing osmotic environment. We present a comprehensive protocol for studying the volume regulatory behavior of chondrocytes within intact cartilage tissue using confocal laser-scanning microscopy. Our data acquisition regime optimizes both signal-to-noise and cell viability during time-lapsed three-dimensional (3-D) (x, y, z, t) imaging. The porcine cartilage is treated as an integrated component of the imaging system, and we demonstrate methods for the direct assessment of tissue-induced axial attenuation and image distortion. Parameterized functions describing these two components of image degradation are used to correct experimental data. The current study also highlights the problems associated with the analysis and visualization of four-dimensional (4-D) images. We have devised two new types of data reconstruction. The first compresses each 3-D time point into a single quantitative view, termed a coordinate view. From these reconstructions we are able to simultaneously view and extract cell measurements. A second type, a 4-D reconstruction, uses color to represent relative changes in cell volume, again while maintaining the morphological and spatial information. Both these approaches of image analysis and visualization have been implemented to study the morphology, spatial distribution, and dynamic volume behavior of chondrocytes after osmotic perturbation. We have mapped chondrocyte shape, arrangement, and absolute volume in situ, which vary significantly from the tissue surface through to the underlying bone. Despite the rigid nature of the extracellular matrix, cartilage cells are osmotically sensitive and respond to stimulation of volume regulatory mechanisms. The combined techniques of confocal laser-scanning microscopy and vital cell labeling have enabled us to study, for the first time, the response of chondrocytes in situ to changes in interstitial osmotic pressure.
面对渗透压应激时，细胞体积调节是一种基本的细胞稳态机制。在正常关节软骨中，软骨细胞暴露于不断变化的渗透环境中。我们提出了一种综合方案，用于使用共聚焦激光扫描显微镜研究完整软骨组织内软骨细胞的体积调节行为。我们的数据采集方案在延时三维（3-D）（x、y、z、t）成像过程中优化了信噪比和细胞活力。猪软骨被视为成像系统的一个集成组件，我们展示了直接评估组织诱导的轴向衰减和图像失真的方法。描述图像退化这两个组成部分的参数化函数用于校正实验数据。当前研究还突出了与四维（4-D）图像分析和可视化相关的问题。我们设计了两种新型数据重建方法。第一种方法将每个3-D时间点压缩成一个单一的定量视图，称为坐标视图。通过这些重建，我们能够同时查看和提取细胞测量数据。第二种类型，即4-D重建，使用颜色来表示细胞体积的相对变化，同时保持形态和空间信息。这两种图像分析和可视化方法均已用于研究渗透扰动后软骨细胞的形态、空间分布和动态体积行为。我们已经绘制了软骨细胞的形状、排列和原位绝对体积，从组织表面到下方骨骼，这些特征差异显著。尽管细胞外基质具有刚性，但软骨细胞对渗透压敏感，并对体积调节机制的刺激做出反应。共聚焦激光扫描显微镜和活细胞标记的联合技术使我们首次能够研究原位软骨细胞对间质渗透压变化的反应。
