We have developed a method for classifying cultured cells on the basis of shape characteristics. High-resolution optical information on thre imensional shape was obtained by anodic oxide interferometry. Each interference order formed in a cell was considered as a closed figure; measurement of 37 mathematical descriptors was carried out for each figure. The individual cells were classified according to the values of their descriptors. We used standard principles of pattern recognition, such as hierarchical cluster analysis and nearest neighbor analysis, as a basis for ordering the cells into groups. Alternatively, linear discriminant functions could be used, but they provided only a slight improvement in correct classification of the cells. We anticipate that the method will be appropriate for classification of cultured cell lines and for determination of the magnitude and direction of cell shape changes implicated in various biological processes. Cell shape is one of the fundamental criteria used for visual classification of cells into histological types. The relationship of cell shape to functional integrity is obvious in some specialized cells (e.g., neurons). Recently, aspects of shape maintenance have also been implicated in mechanisms of growth control in normal cells (1). Other observations have suggested that the shape of cells cultured on solid substrata (2-9), particularly the area of lamellar cytoplasm (2, 3, 7), changes during viral and oncogenic transformation. Research on these problems would be facilitated by methods for quantifying cell shape. Several descriptors of shape have been developed by others for automated cytological diagnosis (10-13). In the present study, we have used many of these descriptors and defined others as the basis of a classification strategy for cultured cells.Besides its application to cultured cells, the present technical approach to the problem of shape analysis differs from that of previous workers in two respects. First, because the threedimensional aspect of cell shape is difficult to evaluate by routine photo-optical methods, we used anodic oxide interference to obtain information on cell height. Because of the multiple reflection characteristics of the interference pattern, this technique also offered better resolving power in the vertical dimension than did traditional light microscopy. The principles of anodic oxide interferometry have been discussed (14, 15) in relation to protein adsorption on model surfaces, including culture substrata. A second area of innovation was in avoiding digitization errors by magnifying the image prior to digitization.We have also employed a large set of shape features for analyzing cells. Frequently, features used for discriminating among biological objects are chosen on the basis of visual discrimination carried out by humans (11-13). We recognized that shape features that might relate to certain aspects of cellular functions would not necessarily be easy to perceive visually (16). Therefore, the approach adopted was to meas...
We have developed a method for permeabilizing CHO cells to nucleotides under conditions which allow most cells to remain viable. Permeabilized cells can carry out ATP-dependent, semiconservative synthesis of DNA. The data are consistent with the continuation of DNA synthesis in those cells in S phase at the time of treatment, possibly limited to completion of replicon synthesis without new initiations.
Autoradiographic patterns of [3H]thymidine incorporation, nuclear/cytoplasmic ratios (N/C), and the percentage of dark epithelial cells were analyzed in a group of epithelial lesions induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rat tracheal transplants. It was found that similar lesions of different age exhibit the same labeling indices (LIs), therefore the lesions of different age were subsequently pooled in the following groups and studied by high resolution light microscopic autoradiography: squamous metaplasia without or with only mild atypia, squamous metaplasia with moderate atypia, squamous metaplasia with severe atypia, carcinoma in situ, and microinvasive carcinoma. Normal tracheal and esophageal epithelia were also analyzed. Whereas the normal tracheal basal layer exhibited an LI smaller than 1%, a clear difference between the carcinomas (in situ and invasive) on one hand (LI approximately 32%) and all the remaining epithelia on the other hand (LI approximately 18%) was detected. The LIs of the suprabasal cells exhibited a statistically significant difference between the squamous epithelia without atypia (LI approximately 2%) and the group comprising all the atypical lesions (LI approximately 9%). Gradients of increasing N/C (nucleus-cytoplasm ratios) values could be observed as the lesions increased in severity, especially in the middle and surface layers (e.g., in the surface layer regular metaplasia N/C = 0.08, squamous metaplasia with moderate atypia N/C = 0.26, and carcinoma in situ N/C = 0.50). Dark cells were absent in the normal esophageal epithelium, were present in moderate numbers in the basal layer of regular squamous metaplasia (18%), and increased markedly in the atypical epithelial lesions (approximately 50% in the atypical squamous metaplasias and 70% in carcinoma in situ). In the suprabasal layer dark cells increased from 3% in squamous metaplasia with moderate atypia to 28% in metaplasia with severe atypia and 56% in carcinoma in situ. The results confirm in a quantitative fashion that disturbances of cell maturation and cell proliferation are key features of dysplastic lesions induced by chemical carcinogens, and suggest the use of objective parameters for evaluation and classification of preneoplastic alterations.
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