Automated analysis of living cells through the quantitative use of automated phase contrast microscopy

A simplified theory of image formation in phase contrast microscopy is presented. It is shown that the phase shift induced in light (related to the refractive index) by the observed object can be reconstructed, point by point, from the phase-contrast digitally sampled image through an appropriate algorithm. This allows one to make quantitative observations on unstained, living cells.     

Quantitative reflection contrast microscopy of living cells

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.     

Automated analysis of living cells through the quantitative use of automated phase contrast microscopy

A simplified theory of image formation in phase contrast microscopy is presented. It is shown that the phase shift induced in light (related to the refractive index) by the observed object can be reconstructed, point by point, from the phase-contrast digitally sampled image through an appropriate algorithm. This allows one to make quantitative observations on unstained, living cells.     

Cell-to-substrate adhesions during spreading and locomotion of carcinoma cells : A study by microcinematography and reflection contrast microscopy

Motility and patterns of adhesion were determined by time-lapse cinematography and reflection contrast microscopy for two types of carcinoma cells, selected for their different motile behavior and not for their malignancy. Cells from the V2 rabbit carcinoma become locomotory soon after having established the necessary contact to the substratum. In contrast, cells from a human epidermoid carcinoma (LICR-OC-1) first attain a fully spread configuration before some cells slightly round up again for a slow locomotory activity of short range and duration. Reflection contrast showed that during spreading and locomotion, the cells from both carcinomas displayed a predominance of grey, the color associated with close contacts. Fully spread cells, on the other hand, presented a multitude of focal contacts in individually different arrangements of black streaks and dots, randomly distributed over the entire cell area. The functional meaning of this heterogeneity in the arrangement of focal contacts in fully spread cells is not yet understood. The importance of close contacts for spreading and locomotion, however, seems to be established and is in agreement with findings reported for other cell types engaged in the same activities. It is therefore suggested that the formation of substrate contacts depends on cellular activity rather than on the cell type.     

Observations on the cytoplasmic membranes of testicular cells,examined by phase contrast and electron microscopy

In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.