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The rapid progress in computer technology makes possible the automatic analysis of thousands of cells on a slide in the field of automatic uterine cancer cytology. Our approach starts with the high-resolution scanning of visually selected and classified single cells determining the training set for discriminant analysis. On the basis of more than 15 morphologic and textural features measured, correct classification results of 95% are reached. Our aim is to speed up our image processing system by means of an Array-Processor, the model AP 120 B from Floating Point Systems. In this study a realistic time estimation of the scanning, segmentation, feature extraction and classification of about 40,000 cells on a slide is performed.

Images of exponentially growing mouse L fibroblasts were used to test whether G1, S, and G2 cells could be discriminated by means of densitometric, geometric, textural and chromatin features. The cells were preincubated with FUdR and labeled with 3H-TdR. The definition of G1, S, and G2 was based on autoradiography used to define labeled S cells, and DNA content used to define unlabeled 2c (G1) and 4c (G2) cells. The methanol-fixed Feulgen-stained nuclei were scanned with a TV Plumbicon camera equipped with an array-processor system. Thirty-one nuclear features were calculated and stored together with the precise coordinates of each cell for later relocation and correlation with the autoradiographic data. The features were geometric, densitometric, textural and chromatin parameters of the Feulgen stained images. Feature evaluation and supervised learning were performed. When DNA content was excluded 14 textural and chromatin features were selected. By using these a correct classification of about 80% was achieved. Inclusion of DNA content and its correlates caused degeneration of performance, yielding 75% correct classification. DNA content alone gave 69% correct classification.

Human lymphocytes, transferred in culture into medium conditioned with phytohemagglutinin, were studied with the combined use of quantitative 14C-uridine autoradiography and high-resolution image analysis. During the first 24 h before the onset of DNA synthesis it was possible to study pure G0 and G1 populations. The return of the proliferating cells into the G0 state was clearly monitored in an RNA synthesis rate/DNA content plot during 1-week growth. On days 3-6, the proliferation decreased progressively, and more cells accumulated in the quiescent G0 state. Geometric, densitometric, and textural parameters evaluated from the Feulgen-stained nuclei of the same cells indicated that the decondensation/condensation processes of the nuclear chromatin during the G0 in equilibrium G1 transitions paralleled the RNA synthesis activity. The results help to associate descriptors of nuclear morphology, as derived from image analysis, with functional parameters during activation and return to quiescence of human lymphocytes.

The nuclear morphology of preneoplastic and unaltered hepatocytes in diethylnitrosamine-treated rats was investigated. Two-micrometer-thick sections of methacrylate-embedded liver were scanned with a TV camera and evaluated in a computer using multivariate analysis methods. The preneoplastic cell populations (islands) were distinguished from unaltered hepatocytes by histochemical demonstration of glycogen storage in specimens from starved animals. After the hemalaun-stained liver sections were scanned randomly, the sections were stained for glycogen, and the previously registered cells were identified visually using a scanning stage for relocation. This objective identification of unaltered and preneoplastic hepatocytes formed the basis for the selection of a training set for feature evaluation and supervised classification. Image analysis for quantitative nuclear morphology was applied to the hemalaun-stained cells. The results showed that condensed chromatin was reduced and nuclear area was increased in the nuclei of glycogen-storing cells. Differences in the nuclear structure were also found. A multivariate analysis including seven features gave a correct classification result of about 82%.

Image analysis was performed using Feulgen-stained smears of L cells that were either confluent or had received a nutritional stimulus to proliferate. The cells were grown directly on slides in order to minimize damage to them during manipulation and to avoid trypsin treatment. A series of experiments was performed by varying the dose and the time of the stimulus. The nuclei were interactively sampled and automatically segmented. Most of the information about the fine chromatin structure was gained after subtracting the original image from a median-filtered image. The results indicate that there were changes in the Feulgen-stained nuclear morphology and densitometry shortly after transition of quiescent G0 cells to the proliferating G1 compartment. Multidimensional cluster analysis provided model-independent discrimination between the resting and the stimulated cells; the latter presented increased nuclear dispersion, indicating increased template activity of the cells. The results obtained are applicable both to obtaining a better understanding of the mechanisms of regulation during the G0-G1 transition and to deriving morphologic criteria applicable in diagnostic routines.

Machine recognition of ectocervical cells has been achieved with good classification success by a number of research groups. Even though a substantial number of cell image features have been introduced, only a moderate number are required for classification. For them, definitions and measures of their discriminatory potential are presented.

Cells of a benign pigmented mole and a malignant melanoma were used to compare electron microscopy (EM) and light microscopy (LM) with high-resolution TV-scanning and multivariate analysis methods. Special emphasis was placed on different kinds of chromatin texture features and their discriminating power. Comparative studies on EM and LM are useful to look for the reliability of light microscopic features for the recognition of malignant melanoma cells. Identical cells of benign pigmented moles and malignant melanomas could be scanned by both EM and LM techniques. As a result, there is a significant correlation between EM and LM texture features, derived from the co-occurrence matrix and the difference statistics histogram. This helps to demonstrate that classification in LM is sufficiently good using texture features only.

The segmentation of scenes of fixed tissue sections for quantitative histopathology is the crucial step for further image processing. Different segmentation methods for the separation of nuclei, nucleoli and whole cells in methacrylate-embedded sections of rat liver were investigated. Reasonable segmentation results were obtained using a contrast-enhanced polar-coordinate transformation to distinguish nuclei, a compactness algorithm to distinguish nucleoli and a skeletonization algorithm to delineate cells when a priori information on the morphometric and photometric properties of the liver tissue was included.

During the passage of erythroblasts down the pathway of maturation, proliferative activity is increasingly slowed and nuclear chromatin increasingly condensed. The aim of the present study was to describe this chromatin condensation by means of textural features and to find correlations between the changes in nuclear structure and the proliferative activity. As a parameter of proliferative activity, the rate of DNA synthesis was investigated in individual cells by utilizing the technique of quantitative 14C-autoradiography. The nuclear fine structure was analyzed in the same cells, after removal of the silver grains and then staining by the Feulgen reaction, by scanning cytophotometry and the application of textural parameters as used in early cancer detection. Among several features, the skewness of the distribution of optical densities proved to be the best parameter for describing the overall morphologic progression during erythroblast maturation. It was almost completely independent of the actual stage of the cells in the cell cycle. The best correlation with the rate of DNA synthesis was found to be the Laplace-filtered optical densities, a parameter that can be interpreted as expressing the "graininess" of chromatin. Since changes in the template activity related to hemoglobinization are also associated with structural changes in chromatin, the relationship between chromatin structure and proliferative activity in erythroblasts is regarded as causal only to a limited extent.