Quantitative reflection contrast microscopy of living cells.

Abstract

Mammalian cells in culture ([baby hamster kidney] BHK-21, [rat kangaroo kidney], Friend [mouse leukemia] human glia and glioma cells) were observed by reflection contrast microscopy. Images of cells photographed at 2 different wavelengths (546 and 436 nm) or at 2 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 influenced by that distance and 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 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 may result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is apparently characterized by a lack of an intermediate layer of culture medium.