Knight M M, Idowu B D, Lee D A, Bader D L
Interdisciplinary Research Centre in Biomedical Materials, Queen Mary, University of London, UK.
Med Biol Eng Comput. 2001 May;39(3):397-404. doi: 10.1007/BF02345297.
This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.
本文研究了在琼脂糖构建体中培养长达7天的分离关节软骨细胞内细胞骨架组织的时间变化。采用荧光标记和共聚焦显微镜观察微管（MT）、波形蛋白中间丝（VIF）和肌动蛋白微丝（AMF）。为了量化细胞群体内细胞骨架组织的程度，开发并全面表征了一种新的图像分析技术。组织程度根据边缘指数进行量化，该指数反映了由Sobel数字滤波器定义的共聚焦图像中“边缘”的密度。该参数被证明与图像强度无关，并且对于所有三种细胞骨架成分，通过组织的视觉评估进行了统计学验证。MT和VIF均表现出贯穿整个细胞质的纤维网络，而AMF则表现为与细胞膜相关的点状单元。边缘指数参数的使用揭示了统计学上显著的时间变化，特别是与VIF和AMF相关。这些发现表明，从完整组织分离后，细胞行为的许多方面可能存在细胞骨架介导的时间变化。此外，图像分析技术可能对未来旨在量化细胞骨架组织变化的研究有用。
