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Methods for numerical description and subsequent classification of cellular protein localization patterns are described. Images representing the localization patterns of 4 proteins and DNA were obtained using fluorescence microscopy and divided into distinct training and test sets. The images were processed to remove out-of-focus and background fluorescence and 2 sets of numeric features were generated: Zernike moments and Haralick texture features. These feature sets were used as inputs to either a classification tree or a neural network. Classifier performance (the average percent of each type of image correctly classified) on previously unseen images ranged from 63% for a classification tree using Zernike moments to 88% for a backpropagation neural network using a combination of features from the 2 feature sets. These results demonstrate the feasibility of applying pattern recognition methods to subcellular localization patterns, enabling sets of previously unseen images from a single class to be classified with an expected accuracy greater than 99%. This will provide not only a new automated way to describe proteins, based on localization rather than sequence, but also has potential application in the automation of microscope functions and in the field of gene discovery.

The Data File Standards Committee of the Society for Analytical Cytology presents a Standard to be used for the storage of data associated with flow cytometric measurements. The Standard specifies a format that provides for the inclusion of all information necessary to fully describe: (1) the instrument used for the measurement; (2) the sample measured; (3) the data obtained; and (4) the results of analysis of the data. The Committee and the Society for Analytical Cytology point out that the use of this Standard by all those individuals and companies that generate or use data taken with flow cytometers or generate methods of analysis for the data will encourage the sharing of such data and methods of analysis.

The nuclear cycle kinetics of Physarum polycephalum plasmodia were examined using flow cytofluorometry. The dyes Hoechst 33342 and propidium iodide were used to stain the DNA of isolated nuclei. In asynchronously growing microplasmodia. S phase consists of 13--15% of the nuclear division cycle time. Nuclei isolated from individual macroplasmodia, which have previously been demonstrated to divide in synchrony, were shown to be less synchronized during late S phase than during mitosis. The results obtained demonstrate the feasibility of flow cytometric measurement of the properties of nuclei isolated from a single cell.

The application of K-means (ISODATA) cluster analysis to flow cytometric data is described. The results of analyses of flow cytometric data for mixtures of fluorescent microspheres and samples of peripheral blood mononuclear cells are presented. A method for simultaneously displaying list mode data for any number of parameters, which had previously been applied to a continuous set of parameters such as multi-angle light scattering data, is used to present the results of cluster analysis on physically unrelated parameters; this method allows rapid evaluation of the success of subpopulation identification. The factors that influence automated identification of subpopulations are examined, and methods for determining optimal values for these factors are described.

Autofluorescence of cells can be a major portion of the fluorescence signal in many systems, especially when fluorescent conjugates are used to study receptor-ligand systems for which there are less than 70,000 receptors per cell. We have devised a method for the cell-by-cell correction of autofluorescence for flow cytometric data by using an additional parameter to measure and correct for autofluorescence in the fluorescence channel. The principle has been extended to allow simultaneous correction for autofluorescence and dual-fluorescence spillover compensation in samples labeled with two different fluorochromes; all corrections were done in software, making them applicable to any flow cytometer. The autofluorescence correction method was used to analyze the acidification of epidermal growth factor (EGF) by Swiss 3T3 cells. EGF is acidified to pH 6.2 starting two min after labeling, with a half-time for acidification of 45 s.

The light-scattering properties of human neutrophils were compared on a cell-by-cell basis before and after stimulation with chemotactic peptide using flow cytometry. Between 20 and 180 sec after peptide addition, side (90 degrees) scatter declined by up to 4% and forward scatter increased up to 6%. Between 3 and 15 min, side scatter increased up to 15% and forward scatter decreased up to 5%. Association of a fluorescence chemoattractant with neutrophils was most rapid during the initial phase of increasing forward and decreasing side scatter, and association saturated before the maximum increase in side scatter. Evidence is presented that the observed changes in scatter were not a consequence of chemoattractant-induced cell-cell adhesion or neutrophil degranulation. Rather, the early phases of light-scattering changes are interpreted to represent membrane ruffling by the stimulated neutrophil; the later phases polarization of the neutrophil morphology.

The increasing complexity of multiparameter data collection and analysis in flow cytometry and the development of relatively inexpensive arc-lamp-based flow cytometers, which increases the probability that laboratories or institutions may have more than one type of instrument, creates a need for shareable analysis programs and for the transport of flow cytometric data files within an installation or from one institution to another. To address this need, we propose a standard file format to be used for all flow cytometric data. The general principles of this proposal are: (1) The data file will contain a minimum of three segments, TEXT, DATA, and ANALYSIS; (2) The TEXT and ANALYSIS segments consist of KEYWORDS, which are the names of data fields, and their values; (3) All TEXT is encoded in ASCII; (4) KEYWORDS and their values may be of any length; (5) Certain KEYWORDS will be standard, i.e., having specified formats to be recognized by all programs. The structure of the DATA segment will be uniquely defined by the values of KEYWORDS in the TEXT area. It may be in any bit resolution, facilitating compatibility between machines with different word length and/or allowing bit compression of the data. The structured nature of the TEXT area should facilitate management of flow cytometric data using existing data base management systems. The proposed file format has been implemented on VAX, PDP-11, and HP9920 based flow cytometry data acquisition systems.

Neoplastic transformation often results in the loss of growth control and concomitant changes in cell surface properties. The changes in endocytosis of a variety of probes after serum or anchorage transformation were measured for mouse fibroblasts by flow cytofluorometry. No major differences in dextran (fluid phase) or histone (nonspecific-adsorptive) endocytosis were observed among four cell lines with different growth properties. However, decreased receptor-mediated internalization of alpha 2-macroglobulin was observed for cell lines transformed to either serum or anchorage independence. Furthermore, increased wheat germ agglutinin and decreased insulin endocytosis were observed, but only in serum transformants. The changes specific to serum transformants were not accounted for by changes in binding of wheat germ agglutinin or insulin. The possible implications of these observations regarding serum transformation and the insulin requirement for growth in serum-free medium are discussed.