In order to measure the nucleation of nouveau adhesions on the ventral surface of a cell, we have combined phase shifting laser feedback interferometry with a high numerical aperture inverted fluorescence microscope. We use fluorescence to image molecules at the adhesion site and stage scanning interference microscopy in order to measure the distance between the ventral surface of a cell and the substratum with several nanometer precision. Our analytic and Monte Carlo simulations of integrin mediated adhesions predict several features of these nouveau adhesions. An analysis of the energetics of membrane bending and the effects of a composite system of freely diffusing repellers and receptors and a fixed network of ligands on the extracellular matrix predicts that a small bundle of actin filaments should be able to push the membrane down to the extracellular matrix and nucleate a nouveau adhesion with critical radius below the diffraction limit. We have obtained a map of the reflectivity of the ventral surface of fixed metastatic mammary adenocarcinoma cells and we have shown that the data are correlated with markers for a focal adhesion adaptor protein. We are modeling the interference of the incident electric field with the field reflected from the ventral surface so as to obtain the surface topography at focal adhesions from the optical phase data.
Keywords: Integrin adhesions, nucleation, critical radius, interference microscopy, phase shifting interferometry
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