Figure 3

SPR and phase contrast images of five cell types (3T3, HepG2, Vero, A10 and A549) fixed under PBS buffer 72 h after plating on fibronectin coated substrates, and SPR image comparison with fluorescently stained α-vinculin. A) SPR images collected with 100X/1.65 NA objective using 590 nm incident light; phase contrast images acquired with a 20X/0.4 NA objective. The SPR image displays distinct changes in reflectivity for various intracellular components within the evanescent wave such as cell membrane, focal adhesions, and cell nucleus. B) SPR image of A10 cell collected as in A) but contrast adjusted on a linear scale to emphasize the focal adhesions. Subsequently, the sample was immunofluorescently labeled with α-vinculin and imaged with a 63X/1.3 NA objective. A manual intensity threshold of α-vinculin was then overlaid onto the SPR image for comparison. C) Imaging conditions same as in A). A region of intermediate intensity, putative extracellular deposited material, is detected along the cell periphery. A linescan in the SPR image is segregated into four distinct image features: fibronectin (FN) coated substratum, extracellular deposited material (ECM), the basal cell surface, and focal adhesions. The scale bar of 25 μm applies to all images in figure.