The closeness of contact between cultured chick heart fibroblasts and glass substrates has been examined by interference reflexion microscopy. Evaluation of the optical technique demonstrated that both the film of medium between the cell and substrate and the cell itself act as two superimposed thin films which generate a complex interference pattern in reflected light or low illuminating numerical aperture (I.N.A.). The interference pattern generated by the cell consists of first and higher orders of interference and can be eliminated from the image by increasing the I.N.A. to greater than 1.0. The residual zero-order interference pattern at high I.N.A. originates from the thin film of medium between cell and substrate and corresponds to the closeness of contact between cell and substrate. Based on the zero-order interference pattern and a comparison of the same cells with differential interference optics, the following features were recognized in living chick heart fibroblasts. Focal contacts, 0.25-0.5 mum wide, 2–10 mum long, separated by 10–15 nm from the substrate, are located under the peripheral regions of the leading lamellae and near the edge of extended non-spreading regions of the cell margin in moving and stationary cells. Individual focal contacts are coincident with cytoplasmic fibres of the same dimensions as the contact or with the peripheral ends of longer fibres that extend from the focal contact toward the centre of the cell. In spreading cells a second type of contact is present under the peripheral regions of the leading lamellae. This close contact consists of a broad area of uniform cell-to-substrate separation distance (ca. 30 nm). Focal contacts are distributed within the close contact under the leading lamellae. Lamellipodia extend ahead of the close contact and are separated by 100 nm or more from the substrate. Depending on the previous motile history of the cell, the close contact extends to varying degrees under the centre of the cell but it is typically dissected here by discrete areas of distinctly greater cell-to-substrate separation (100-140 nm). Evidence for the adhesive nature of the focal contacts is considered. The common identity is discussed of the focal contacts and associated cytoplasmic fibres described here in living cells with the regions of closest apposition to the substrate and associated cytoplasmic plaques and bundles of microfilaments seen in EM studies.

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