Microinterferometry of the movement of dry matter in fibroblasts

Abstract

We describe the use of interferometric microscopy coupled with a novel application of Senarmont compensation for detecting and quantifying the distribution of dry matter in cultured cells. In conjunction with video techniques and digital image processing, a two-dimensional, calibrated map of the dry mass distribution in an isolated cell can be obtained and digitally recorded. We have called the technique Digitally Recorded Interferometric Microscopy with Analyser Shift (DRIMAS). The method greatly facilitates the automatic recognition of cells by computer. Recorded time-lapse sequences can be used to establish a database of the growth and motility of specific cells in given experimental conditions. Databases of this type can be analysed to reveal the patterns of growth and locomotory behaviour of individual cells. We describe a systematic method of obtaining parameters of cell size, shape, spreading, intracellular motility and translocation. Auto-correlations and cross-correlations between these parameters can be detected and quantified using time series analysis, revealing potential cause/effect relationships in the mechanisms of growth and motility. Besides characterizing the overall pattern of cell behaviour, these data can also yield information about the instantaneous pattern of intracellular motility. We describe the use of finite element analysis to reveal the dynamics of the intracellular transport of dry matter. This yields the pattern of the minimum flow of dry matter required to account for the changes in its distribution. Most of this flux is not associated with the movement of visible structures and possibly represents the transport of dissociated components of the cytoskeleton. In chick heart fibroblasts, surprisingly high velocities of nearly 2.0 microns s-1 were detected during the period of increased motility following tail detachment. The total kinetic energy associated with the dry mass flux is a single parameter, which characterizes the instantaneous motility of the cell. We found that the kinetic energy of intracellular motility can be several hundred times greater than the kinetic energy of translocation. Kinetic energy may prove to be a very informative single measure of intracellular motility for assessing the effects of malignant transformation, genetic manipulations, and other experimental treatments on the locomotory machinery of the cell.