Interference reflection microscopy in cell biology: methodology and applications

Abstract

Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.