Phase-shifting interference microscopy applied to the analysis of cell behaviour.

The theory of phase-shifting interferometry is not new but it is only recently, with the advent of solid-state detector arrays and fast image processors, that it has become a practical imaging technique. In conjunction with transmission interference microscopy, phase-shifting presents a new way of introducing contrast into the images of transparent microscopic objects such as cultured cells. An earlier paper from our laboratory has emphasised the advantages of transmission interference microscopy over phase contrast or differential interference contrast microscopy for the computerised analysis of cell behaviour. Phase-shifting greatly improves the accuracy, long-term stability and range of application of this technique but it has not previously, to our knowledge, been combined with transmission interference microscopy for the study of cultured cells. The resulting image is especially well suited to quantitative analysis by computer since it is a direct representation of the distribution of non-aqueous cellular material in the specimen. The image is not degraded by uneven illumination; by heterogeneous sensitivity of the detector array; or by differential absorption of light in the optics or specimen. Our main purpose in developing the method is to obtain sequences of images of the motile behaviour of cells in culture for analysis by computer. This type of analysis is potentially a powerful tool for studying the motile responses of cells and the operation and control of their locomotory machinery. Not only can the method be used for studying cell translocation and the dynamics of intracellular movement of non-aqueous material, but it is now possible to study in detail the time course of growth in individual cultured cells.